Abstract:

The invention provides vitamin D3 analogs of cholecalciferol,
substituted at carbon 20 with cycloalkyl, e.g., cyclopropyl, wherein
carbon-16 is a double bond, and carbon-23 is a single, double, or triple
bond. Various alkyl or haloalkyl substitutions are incorporated as
carbon-25. The invention provides pharmaceutically acceptable esters,
salts, and prodrugs thereof. Methods for using the compounds to treat
vitamin D3 associated states, and pharmaceutical compositions
containing the compounds are also disclosed.

Claims:

1. A vitamin D3 compound of formula I:wherein:B is single, double, or
triple bond;X1 and X2 are each independently H2 or
CH2, provided X1 and X2 are not both CH2;R1 is
hydroxyl or halogen;R2 and R3 taken together with C20 form
C3-C6 cycloalkyl;R4 and R5 are each independently
alkyl, or haloalkyl;R6 is hydrogen, C1-C4 alkyl,
hydroxyalkyl, or haloalkyl, with the understanding that R6 is absent
when B is a triple bond; andpharmaceutically acceptable esters, salts,
and prodrugs thereof.

2. The compound of claim 1, wherein R1 is hydroxyl.

3. The compound of claim 1, wherein R1 is halogen.

4. The compound of claim 3, wherein R1 is F.

5. The compound of claim 1, wherein B is a single bond.

6. The compound of claim 1, wherein B is a double bond.

7. The compound of claim 1, wherein B is a triple bond.

8. The compound of claim 1, wherein X1 is CH2 and X2 is
H.sub.2.

9. The compound of claim 1, wherein X1 and X2 are each H.sub.2.

10. The compound of claim 1, wherein R4 and R5 are each
independently alkyl, or haloalkyl.

11. The compound of claim 1, wherein R4 and R5 are each
independently alkyl, or trihaloalkyl.

12. The compound of claim 1, wherein R4 and R5 are each
independently methyl, or trifluoromethyl.

13. The compound of claim 1, wherein R4 and R5 are methyl.

14. The compound of claim 1, wherein R4 and R5 are
trifluoromethyl.

15. The compound of claim 1, wherein R6 is hydrogen.

16. The compound of claim 1, wherein R2 and R3 taken together
with C20 form cyclopropyl.

17. The compound of claim 1 having the formula I-awherein:B is single,
double, or triple bond;X1 and X2 are each independently H2
or CH2, provided X1 and X2 are not both CH2;
andR4 and R5 are each independently alkyl, or haloalkyl.

18. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-23-yne-20-cyclopyl-cholecalciferol:

19. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-23-yne-20-cyclopropyl-19-nor-cholecalciferol:

20. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-19-nor-chole-
calciferol:

21. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-cholecalcife-
rol:

22. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-cholec-
alciferol:

23. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-cholecalcifer-
ol:

24. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-cholec-
alciferol:

25. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-cholecalcifer-
ol:

26. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecalciferol:

27. The compound of claim 17, wherein said compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

28. The compound of claim 1 having the formula I-bwherein:B is single,
double, or triple bond;X1 and X2 are each independently H2
or CH2, provided X1 and X2 are not both CH2;
andR4 and R5 are each independently alkyl, or haloalkyl.

29. The compound of claim 28, wherein said compound is
1.alpha.-Fluoro-25-hydroxy-16-ene-23-yne-20-cyclopropyl-cholecalciferol:

30. The compound of claim 28, wherein said compound is
1.alpha.-Fluoro-25-hydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro--
cholecalciferol:

31. The compound of claim 28, wherein said compound is
1.alpha.-Fluoro-25-hydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-c-
holecalciferol:

32. The compound of claim 28, wherein said compound is
1.alpha.-Fluoro-25-hydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-c-
holecalciferol:

33. A method for treating a subject for a vitamin D3 associated
state, comprising administering to said subject in need thereof an
effective amount of a vitamin D3 compound formula I:wherein:B is
single, double, or triple bond;X1 and X2 are each independently
H2 or CH2, provided X1 and X2 are not both
CH2;R1 is hydroxyl or halogen;and R1 taken together with
C20 form C3-C6 cycloalkyl;R4 and R5 are each
independently alkyl, or haloalkyl;R6 is hydrogen, C1-C4
alkyl, hydroxyalkyl, or haloalkyl, with the understanding that R6 is
absent when B is a triple bond; andpharmaceutically acceptable esters,
salts, and prodrugs thereof, such that said subject is treated for said
vitamin D3 associated state.

34. The method according to claim 33, further comprising the step of
obtaining the vitamin D compound.

35. The method of claim 33, further comprising identifying a subject in
need of treatment for a vitamin D3 associated state.

36. The method of claim 33, wherein said vitamin D3 associated state
is selected from the group consisting of an ILT3-associated disorder and
a disorder characterized by an aberrant activity of a vitamin
D3-responsive cell.

37-42. (canceled)

43. The method of claim 36, wherein said disorder characterized by an
aberrant activity of a vitamin D3-responsive cell is selected from the
group consisting of a disorder characterized by an aberrant activity of a
hyperproliferative skin cell, a disorder characterized by an aberrant
activity of an endocrine cell, a disorder characterized by an aberrant
activity of a bone cell, a disorder is characterized by an aberrant
activity of a vitamin D3-responsive smooth muscle cell, cirrhosis,
chronic renal disease, hypertension, benign prostate hypertrophy,
neoplastic disease, neuronal loss, uveitis and interstitial cystitis.

44. The method of claim 43, wherein said disorder characterized by an
aberrant activity of a hyperproliferative skin cell is psoriasis.

45-51. (canceled)

52. The method of claim 51, wherein said disorder characterized by an
aberrant activity of a bone cell is osteoporosis.

53. (canceled)

54. The method of claim 52, wherein the Vitamin D3 compound has the
formula I-awherein:B is single, double, or triple bond;X1 and
X2 are each independently H2 or CH2, provided X1 and
X2 are not both CH2; andR4 and R5 are each
independently alkyl, or haloalkyl.

55. The method of claim 54, wherein said vitamin D3 compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

56. The method of claim 54, wherein said compound is
1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-cholec-
alciferol:

57-78. (canceled)

79. A method of ameliorating a deregulation of calcium and phosphate
metabolism, comprising administering to a subject a therapeutically
effective amount of a compound of claim 1, so as to ameliorate the
deregulation of the calcium and phosphate metabolism.

80. The method of claim 79, wherein the deregulation of the calcium and
phosphate metabolism leads to osteoporosis.

81. A method of modulating the expression of an immunoglobulin-like
transcript 3 (ILT3) surface molecule in a cell, comprising contacting
said cell with a compound of claim 1, in an amount effective to modulate
the expression of an immunoglobulin-like transcript 3 (ILT3) surface
molecule in said cell.

82-86. (canceled)

87. A method of inducing immunological tolerance in a subject, comprising
administering to said subject a compound of claim 1, in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
inducing immunological tolerance in said subject.

88-89. (canceled)

90. A method of inhibiting transplant rejection in a subject comprising
administering to said subject a compound of claim 1, in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
inhibiting transplant rejection in said subject.

91-93. (canceled)

94. A method for modulating immunosuppressive activity by an
antigen-presenting cell, comprising contacting an antigen-presenting cell
with a compound of claim 1, in an amount effective to modulate ILT3
surface molecule expression, thereby modulating said immunosuppressive
activity by said antigen-presenting cell.

95-96. (canceled)

97. A method for preventing or treating bladder dysfunction in a subject
in need thereof by administering an effective amount of a compound of
claim 1, thereby to prevent or treat bladder dysfunction in said subject.

98-102. (canceled)

103. The method of claim 33, wherein said vitamin D3 compound is
administered in combination with a pharmaceutically acceptable carrier or
diluent.

104. The method of claim 103, wherein said vitamin D3 compound is
administered to the subject using a pharmaceutically-acceptable
formulation.

105. The method of claim 104, wherein said pharmaceutically-acceptable
formulation provides sustained delivery of said vitamin D3 compound
to a subject for at least four weeks after the
pharmaceutically-acceptable formulation is administered to the subject.

106-108. (canceled)

109. The method of claim 33, wherein the subject is a mammal.

110. The method of claim 109, wherein the subject is human.

111. The method of claim 33, wherein said compound is administered orally,
intravenously, topically or parenterally.

112-114. (canceled)

115. The method claim 33, wherein said compound is administered at a
concentration of 0.001 μg-100 μg/kg of body weight.

116. A pharmaceutical composition comprising an effective amount of a
compound of formula I:wherein:B is single, double, or triple bond;X1
and X2 are each independently H2 or CH2, provided X1
and X2 are not both CH2;R1 is hydroxyl or halogen;R2
and R taken together with C20 form C1-C6
cycloalkyl;R4 and R5 are each independently alkyl, or
haloalkyl:R6 is hydrogen, C1-C4 alkyl, hydroxyalkyl, or
haloalkyl, with the understanding that R6 is absent when B is a
triple bond; andpharmaceutically acceptable esters, salts, and prodrugs
thereof, and a pharmaceutically acceptable diluent or carrier.

117. The pharmaceutical composition of claim 116, wherein said effective
amount is effective to treat a vitamin D3 associated state.

118-121. (canceled)

122. A packaged formulation for use in the treatment of a vitamin D3
associated state, comprising a pharmaceutical composition comprising a
compound of claim 1, and instructions for use in the treatment of a
vitamin D3 associated state.

123-125. (canceled)

Description:

RELATED APPLICATION

[0001]This application claims priority to U.S. provisional patent
application Ser. No. 60/612,732, filed Sep. 24, 2004, the disclosure of
which is incorporated herein in its entirety by this reference.

BACKGROUND OF THE INVENTION

[0002]The importance of vitamin D (cholecalciferol) in the biological
systems of higher animals has been recognized since its discovery by
Mellanby in 1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council
(GB) SRS 61:4). It was in the interval of 1920-1930 that vitamin D
officially became classified as a "vitamin" that was essential for the
normal development of the skeleton and maintenance of calcium and
phosphorous homeostasis.

[0005]Vitamin D3 and its hormonally active forms are well-known
regulators of calcium and phosphorous homeostasis. These compounds are
known to stimulate, at least one of, intestinal absorption of calcium and
phosphate, mobilization of bone mineral, and retention of calcium in the
kidneys. Furthermore, the discovery of the presence of specific vitamin D
receptors in more than 30 tissues has led to the identification of
vitamin D3 as a pluripotent regulator outside its classical role in
calcium/bone homeostasis. A paracrine role for 1α,25(OH)2
D3 has been suggested by the combined presence of enzymes capable of
oxidizing vitamin D3 into its active forms, e.g.,
25-OHD-1α-hydroxylase, and specific receptors in several tissues
such as bone, keratinocytes, placenta, and immune cells. Moreover,
vitamin D3 hormone and active metabolites have been found to be
capable of regulating cell proliferation and differentiation of both
normal and malignant cells (Reichel, H. et al. (1989) Ann. Rev. Med. 40:
71-78).

[0006]Given the activities of vitamin D3 and its metabolites, much
attention has focused on the development of synthetic analogs of these
compounds. A large number of these analogs involve structural
modifications in the A ring, B ring, C/D rings, and, primarily, the side
chain (Bouillon, R. et al., Endocrine Reviews 16(2):201-204). Although a
vast majority of the vitamin D3 analogs developed to date involve
structural modifications in the side chain, a few studies have reported
the biological profile of A-ring diastereomers (Norman, A. W. et al. J.
Biol. Chem. 268 (27): 20022-20030). Furthermore, biological
esterification of steroids has been studied (Hochberg, R. B., (1998)
Endocr Rev. 19(3): 331-348), and esters of vitamin D3 are known (WO
97/11053).

[0007]Moreover, despite much effort in developing synthetic analogs,
clinical applications of vitamin D and its structural analogs have been
limited by the undesired side effects elicited by these compounds after
administration to a subject for known indications/applications of vitamin
D compounds.

SUMMARY OF THE INVENTION

[0008]The invention is directed to vitamin D3 compounds of the
formula:

wherein: B is a single, double, or triple bond; X1 and X2 are
each independently H2 or CH2, provided X1 and X2 are
not both CH2; R1 is hydroxyl or halogen; R2, R3 and
R6 are each independently hydrogen, C1-C4 alkyl,
hydroxyalkyl, or haloalkyl, with the understanding that R6 is absent
when B is a triple bond, or R2 and R3 taken together with
C20 form C3-C6 cycloalkyl; R4 and R5 are each
independently alkyl or haloalkyl; and pharmaceutically acceptable esters,
salts, and prodrugs thereof.

[0009]Thus in one aspect, the invention provides a vitamin D3
compound of formula I:

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2;R1 is hydroxyl or halogen;R2 and R3 taken
together with C20 form C3-C6 cycloalkyl;R4 and
R5 are each independently alkyl, or haloalkyl;R6 is hydrogen,
C1-C4 alkyl, hydroxyalkyl, or haloalkyl, with the understanding
that R6 is absent when B is a triple bond; andpharmaceutically
acceptable esters, salts, and prodrugs thereof.

[0010]In another aspect, the method provides a method of ameliorating a
deregulation of calcium and phosphate metabolism. The method includes
administering to a subject a therapeutically effective amount of a
vitamin D3 compound of formula I, so as to ameliorate the
deregulation of the calcium and phosphate metabolism.

[0011]In another aspect, the invention provides a method of modulating the
expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule
in a cell. The method includes contacting the cell with a vitamin D3
compound of formula I in an amount effective to modulate the expression
of an immunoglobulin-like transcript 3 (ILT3) surface molecule in the
cell.

[0012]In yet another aspect, the invention provides a method of treating
an ILT3-associated disorder in a subject. The method includes
administering to the subject a vitamin D3 compound of formula I in
an amount effective to modulate the expression of an ILT3 surface
molecule, thereby treating the ILT3-associated disorder in the subject.

[0013]In still another aspect, the invention provides a method of inducing
immunological tolerance in a subject. The method includes administering
to the subject a vitamin D3 compound of formula I in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
inducing immunological tolerance in the subject.

[0014]In a further aspect, the invention provides a method of inhibiting
transplant rejection in a subject. The method includes administering to
the subject a vitamin D3 compound of formula I in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
inhibiting transplant rejection in the subject.

[0015]In still another embodiment, the invention provides a method for
preventing or treating bladder dysfunction in a subject in need thereof
by administering an effective amount of a vitamin D3 compound of
formula I thereby to prevent or treat bladder dysfunction in said
subject.

[0016]In yet another aspect, the invention provides a packaged formulation
for use in the treatment of a vitamin D3 associated state. The
packaged formulation includes a pharmaceutical composition comprising a
vitamin D3 compound of formula I and a pharmaceutically-acceptable
carrier, packaged with instructions for use in the treatment of a vitamin
D3 associated state.

[0017]In another aspect, the invention provides a packaged formulation for
use in the treatment of an ILT-3 associated disorder. The packed
formulation includes a pharmaceutical composition comprising a vitamin
D3 compound of formula I and a pharmaceutically-acceptable carrier,
packaged with instructions for use in the treatment of an ILT3-associated
disorder.

[0018]In a further aspect, the invention provides a method for modulating
immunosuppressive activity by an antigen-presenting cell. The method
includes contacting an antigen-presenting cell with a vitamin D3
compound of formula I in an amount effective to modulate ILT3 surface
molecule expression, thereby modulating the immunosuppressive activity by
the antigen-presenting cell.

[0019]In yet another aspect, the invention provides a pharmaceutical
composition. The composition comprises an effective amount of a vitamin
D3 compound of formula I, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]The present invention is further described below with reference to
the following non-limiting examples and with reference to the following
figures, in which:

[0025]Before further description of the present invention, and in order
that the invention may be more readily understood, certain terms are
first defined and collected here for convenience.

[0026]The term "administration" or "administering" includes routes of
introducing the vitamin D3 compound(s) to a subject to perform their
intended function. Examples of routes of administration which can be used
include injection (subcutaneous, intravenous, parenterally,
intraperitoneally, intrathecal), oral, inhalation, rectal and
transdermal. The pharmaceutical preparations are, of course, given by
forms suitable for each administration route. For example, these
preparations are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration by
injection, infusion or inhalation; topical by lotion or ointment; and
rectal by suppositories. Oral administration is preferred. The injection
can be bolus or can be continuous infusion. Depending on the route of
administration, the vitamin D3 compound can be coated with or
disposed in a selected material to protect it from natural conditions
which may detrimentally effect its ability to perform its intended
function. The vitamin D3 compound can be administered alone, or in
conjunction with either another agent as described above or with a
pharmaceutically-acceptable carrier, or both. The vitamin D3
compound can be administered prior to the administration of the other
agent, simultaneously with the agent, or after the administration of the
agent. Furthermore, the vitamin D3 compound can also be administered
in a proform which is converted into its active metabolite, or more
active metabolite in vivo.

[0027]The term "alkyl" refers to the radical of saturated aliphatic
groups, including straight-chain alkyl groups, branched-chain alkyl
groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. The term alkyl further
includes alkyl groups, which can further include oxygen, nitrogen, sulfur
or phosphorous atoms replacing one or more carbons of the hydrocarbon
backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In
preferred embodiments, a straight chain or branched chain alkyl has 30 or
fewer carbon atoms in its backbone (e.g., C1-C30 for straight
chain, C3-C30 for branched chain), preferably 26 or fewer, and
more preferably 20 or fewer, and still more preferably 4 or fewer.
Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring
structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring
structure.

[0028]Moreover, the term alkyl as used throughout the specification and
claims is intended to include both "unsubstituted alkyls" and
"substituted alkyls," the latter of which refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,
imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will
be understood by those skilled in the art that the moieties substituted
on the hydrocarbon chain can themselves be substituted, if appropriate.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" moiety is an alkyl substituted with an
aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls described above, but that contain at least one
double or triple bond respectively.

[0029]Unless the number of carbons is otherwise specified, "lower alkyl"
as used herein means an alkyl group, as defined above, but having from
one to ten carbons, more preferably from one to six, and most preferably
from one to four carbon atoms in its backbone structure, which may be
straight or branched-chain. Examples of lower alkyl groups include
methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and
so forth. In preferred embodiment, the term "lower alkyl" includes a
straight chain alkyl having 4 or fewer carbon atoms in its backbone,
e.g., C1-C4 alkyl.

[0030]The terms "alkoxyalkyl," "polyaminoalkyl" and "thioalkoxyalkyl"
refer to alkyl groups, as described above, which further include oxygen,
nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon
backbone, e.g., oxygen, nitrogen or sulfur atoms.

[0031]The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic
groups analogous in length and possible substitution to the alkyls
described above, but that contain at least one double or triple bond,
respectively. For example, the invention contemplates cyano and propargyl
groups.

[0032]The term "antigen" includes a substance which elicits an immune
response. The antigens of the invention to which tolerance is induced may
or may not be exogenously derived relative to the host. For example, the
method of the invention may be used to induce tolerance to an
"autoantigen." An autoantigen is a normal constituent of the body that
reacts with an autoantibody. The invention also includes inducing
tolerance to an "alloantigen." Alloantigen refers to an antigen found
only in some members of a species, for example the blood group
substances. An allograft is a graft to a genetically different member of
the same species. Allografts are rejected by virtue of the immunological
response of T lymphocytes to histocompatibility antigens. The method of
the invention also provides for inducing tolerance to a "xenoantigen."
Xenoantigens are substances that cause an immune reaction due to
differences between different species. Thus, a xenograft is a graft from
a member of one species to a member of a different species. Xenografts
are usually rejected within a few days by antibodies and cytotoxic T
lymphocytes to histocompatibility antigens.

[0033]The language "antigen-presenting cell" or "APC" includes a cell that
is able to present an antigen to, for example, a T helper cell.
Antigen-presenting cells include B lymphocytes, accessory cells or
non-lymphocytic cells, such as dendritic cells, Langerhans cells, and
mononuclear phagocytes that help in the induction of an immune response
by presenting antigen to helper T lymphocytes. The antigen-presenting
cell of the present invention is preferably of myeloid origin, and
includes, but is not limited to, dendritic cells, macrophages, monocytes.
APCs of the present invention may be isolated from the bone marrow,
blood, thymus, epidermis, liver, fetal liver, or the spleen.

[0034]The terms "antineoplastic agent" and "antiproliferative agent" are
used interchangeably herein and includes agents that have the functional
property of inhibiting the proliferation of a vitamin D3-responsive
cells, e.g., inhibit the development or progression of a neoplasm having
such a characteristic, particularly a hematopoietic neoplasm.

[0035]The term "aryl" as used herein, refers to the radical of aryl
groups, including 5- and 6-membered single-ring aromatic groups that may
include from zero to four heteroatoms, for example, benzene, pyrrole,
furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole,
tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and
the like. Aryl groups also include polycyclic fused aromatic groups such
as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having
heteroatoms in the ring structure may also be referred to as "aryl
heterocycles," "heteroaryls" or "heteroaromatics." The aromatic ring can
be substituted at one or more ring positions with such substituents as
described above, as for example, halogen, hydroxyl, alkoxy,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino
(including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also
be fused or bridged with alicyclic or heterocyclic rings which are not
aromatic so as to form a polycycle (e.g., tetralin).

[0038]By "bladder dysfunction" is meant bladder conditions associated with
overactivity of the detrusor muscle, for example, clinical BPH or
overactive bladder. In the context of the present invention "bladder
dysfunction" excludes bladder cancer.

[0039]The language "bone metabolism" includes direct or indirect effects
in the formation or degeneration of bone structures, e.g., bone
formation, bone resorption, etc., which may ultimately affect the
concentrations in serum of calcium and phosphate. This term is also
intended to include effects of compounds of the invention in bone cells,
e.g., osteoclasts and osteoblasts, that may in turn result in bone
formation and degeneration.

[0040]The language "calcium and phosphate homeostasis" refers to the
careful balance of calcium and phosphate concentrations, intracellularly
and extracellularly, triggered by fluctuations in the calcium and
phosphate concentration in a cell, a tissue, an organ or a system.
Fluctuations in calcium levels that result from direct or indirect
responses to compounds of the invention are intended to be included by
these terms.

[0041]The term "carcinoma" is art recognized and refers to malignancies of
epithelial or endocrine tissues including respiratory system carcinomas,
gastrointestinal system carcinomas, genitourinary system carcinomas,
testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine
system carcinomas, and melanomas. Exemplary carcinomas include those
forming from tissue of the cervix, lung, prostate, bladder, breast, head
and neck, colon and ovary. The term also includes carcinosarcomas, e.g.,
which include malignant tumors composed of carcinomatous and sarcomatous
tissues.

[0042]An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.

[0043]The term "chiral" refers to molecules which have the property of
non-superimposability of the mirror image partner, while the term
"achiral" refers to molecules which are superimposable on their mirror
image partner.

[0044]The term "diastereomers" refers to stereoisomers with two or more
centers of dissymmetry and whose molecules are not mirror images of one
another.

[0045]The term "effective amount" includes an amount effective, at dosages
and for periods of time necessary, to achieve the desired result, e.g.,
sufficient treat a vitamin D3 associated state or to modulate ILT3
expression in a cell. An effective amount of vitamin D3 compound may
vary according to factors such as the disease state, age, and weight of
the subject, and the ability of the vitamin D3 compound to elicit a
desired response in the subject. Dosage regimens may be adjusted to
provide the optimum therapeutic response. An effective amount is also one
in which any toxic or detrimental effects (e.g., side effects) of the
vitamin D3 compound are outweighed by the therapeutically beneficial
effects.

[0046]A therapeutically effective amount of vitamin D3 compound
(i.e., an effective dosage) may range from about 0.001 to 30 μg/kg
body weight, preferably about 0.01 to 25 μg/kg body weight, more
preferably about 0.1 to 20 μg/kg body weight, and even more preferably
about 1 to 10 μg/kg, 2 to 9 μg/kg, 3 to 8 μg/kg, 4 to 7
μg/kg, OT 5 to 6 μg/kg body weight. The skilled artisan will
appreciate that certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the severity of
the disease or disorder, previous treatments, the general health and/or
age of the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a vitamin D3
compound can include a single treatment or, preferably, can include a
series of treatments. In one example, a subject is treated with a vitamin
D3 compound in the range of between about 0.1 to 20 μg/kg body
weight, one time per week for between about 1 to 10 weeks, preferably
between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and
even more preferably for about 4, 5, or 6 weeks. It will also be
appreciated that the effective dosage of a vitamin D3 compound used
for treatment may increase or decrease over the course of a particular
treatment.

[0047]The term "enantiomers" refers to two stereoisomers of a compound
which are non-superimposable mirror images of one another. An equimolar
mixture of two enantiomers is called a "racemic mixture" or a "racemate."

[0048]The language "genomic" activities or effects of vitamin D3 is
intended to include those activities mediated by the nuclear receptor for
1α, 25(OH)2D3 (VD3R), e.g., transcriptional
activation of target genes.

[0049]The term "haloalkyl" is intended to include alkyl groups as defined
above that are mono-, di- or polysubstituted by halogen, e.g.,
fluoromethyl and trifluoromethyl.

[0050]The term "halogen" designates --F, --Cl, --Br or --I.

[0051]The term "hydroxyl" means --OH.

[0052]The term "heteroatom" as used herein means an atom of any element
other than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.

[0053]The term "homeostasis" is art-recognized to mean maintenance of
static, or constant, conditions in an internal environment.

[0054]The language "hormone secretion" is art-recognized and includes
activities of vitamin D3 compounds that control the transcription
and processing responsible for secretion of a given hormone e.g., a
parathyroid hormone (PTH) of a vitamin D3 responsive cell (Bouillon,
R. et al. (1995) Endocrine Reviews 16(2):235-237).

[0055]The language "hypercalcemia" or "hypercalcemic activity" is intended
to have its accepted clinical meaning, namely, increases in calcium serum
levels that are manifested in a subject by the following side effects,
depression of central and peripheral nervous system, muscular weakness,
constipation, abdominal pain, lack of appetite and, depressed relaxation
of the heart during diastole. Symptomatic manifestations of hypercalcemia
are triggered by a stimulation of at least one of the following
activities, intestinal calcium transport, bone calcium metabolism and
osteocalcin synthesis (reviewed in Boullion, R. et al. (1995)
Endocrinology Reviews 16(2): 200-257).

[0056]The terms "hyperproliferative" and "neoplastic" are used
interchangeably, and include those cells having the capacity for
autonomous growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and neoplastic
disease states may be categorized as pathologic, i.e., characterizing or
constituting a disease state, or may be categorized as non-pathologic,
i.e., a deviation from normal but not associated with a disease state.
The term is meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells, tissues,
or organs, irrespective of histopathologic type or stage of invasiveness.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated with
wound repair.

[0057]The language "immunoglobulin-like transcript 3" or "ILT3" refers to
a cell surface molecule of the immunoglobulin superfamily, which is
expressed by antigen-presenting cells (APCs) such as monocytes,
macrophages and dendritic cells. ILT3 is a member of the
immunoglobulin-like transcript (ILT) family and displays a long
cytoplasmic tail containing putative immunoreceptor tyrosine-based
inhibitory motifs (ITIMs). ILT3 has been shown to behave as an inhibitory
receptor when cross-linked to a stimulatory receptor. A cytoplasmic
component of the ILT3-mediated signaling pathway is the SH2-containing
phosphatase SHP-1, which becomes associated with ILT3 upon cross-linking.
ILT3 is also internalized and ILT3 ligands are efficiently presented to
specific T cells (see, e.g., Cella, M. et al. (1997) J. Exp. Med.
185:1743). The determination of whether the candidate vitamin D3
compound modulates the expression of the ILT3 surface molecule can be
accomplished, for example, by comparison of ILT3 surface molecule
expression to a control, by measuring mRNA expression, or by measuring
protein expression.

[0059]The term "immune response" includes T and/or B cell responses, e.g.,
cellular and/or humoral immune responses. The claimed methods can be used
to reduce both primary and secondary immune responses. The immune
response of a subject can be determined by, for example, assaying
antibody production, immune cell proliferation, the release of cytokines,
the expression of cell surface markers, cytotoxicity, and the like.

[0060]The terms "immunological tolerance" or "tolerance to an antigen" or
"immune tolerance" include unresponsiveness to an antigen without the
induction of a prolonged generalized immune deficiency. Consequently,
according to the invention, a tolerant host is capable of reacting to
antigens other than the tolerizing antigen. Tolerance represents an
induced depression in the response of a subject that, had it not been
subjected to the tolerance-inducing procedure, would be competent to
mount an immune response to that antigen. In one embodiment of the
invention, immunological tolerance is induced in an antigen-presenting
cell, e.g., an antigen-presenting cell derived from the myeloid or
lymphoid lineage, dendritic cells, monocytes and macrophages.

[0061]The language "immunosuppressive activity" refers to the process of
inhibiting a normal immune response. Included in this response is when T
and/or B clones of lymphocytes are depleted in size or suppressed in
their reactivity, expansion or differentiation. Immunosuppressive
activity may be in the form of inhibiting or blocking an immune response
already in progress or may involve preventing the induction of an immune
response. The functions of activated T cells may be inhibited by
suppressing immune cell responses or by inducing specific tolerance, or
both. Immunosuppression of T cell responses is generally an active,
non-antigen-specific, process that requires continuous exposure of the T
cells to the suppressive agent. Tolerance, which involves inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists
after exposure to the tolerizing agent has ceased. Operationally,
tolerance can be demonstrated by the lack of a T cell response upon
re-exposure to specific antigen in the absence of the tolerizing agent.

[0062]The language "improved biological properties" refers to any activity
inherent in a compound of the invention that enhances its effectiveness
in vivo. In a preferred embodiment, this term refers to any qualitative
or quantitative improved therapeutic property of a vitamin D3
compound, such as reduced toxicity, e.g., reduced hypercalcemic activity.

[0063]The language "inhibiting the growth" of the neoplasm includes the
slowing, interrupting, arresting or stopping its growth and metastases
and does not necessarily indicate a total elimination of the neoplastic
growth.

[0065]The term "isomers" or "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.

[0066]The term "leukemia" is intended to have its clinical meaning,
namely, a neoplastic disease in which white corpuscle maturation is
arrested at a primitive stage of cell development. The disease is
characterized by an increased number of leukemic blast cells in the bone
marrow, and by varying degrees of failure to produce normal hematopoietic
cells. The condition may be either acute or chronic. Leukemia's are
further typically categorized as being either lymphocytic i.e., being
characterized by cells which have properties in common with normal
lymphocytes, or myelocytic (or myelogenous), i.e., characterized by cells
having some characteristics of normal granulocytic cells. Acute
lymphocytic leukemia ("ALL") arises in lymphoid tissue, and ordinarily
first manifests its presence in bone marrow. Acute myelocytic leukemia
("AML") arises from bone marrow hematopoietic stem cells or their
progeny. The term acute myelocytic leukemia subsumes several subtypes of
leukemia: myeloblastic leukemia, promyelocytic leukemia, and
myelomonocytic leukemia. In addition, leukemias with erythroid or
megakaryocytic properties are considered myelogenous leukemias as well.

[0067]The term "leukemic cancer" refers to all cancers or neoplasias of
the hemopoietic and immune systems (blood and lymphatic system). The
acute and chronic leukemias, together with the other types of tumors of
the blood, bone marrow cells (myelomas), and lymph tissue (lymphomas),
cause about 10% of all cancer deaths and about 50% of all cancer deaths
in children and adults less than 30 years old. Chronic myelogenous
leukemia (CML), also known as chronic granulocytic leukemia (CGL), is a
neoplastic disorder of the hematopoietic stem cell. The term "leukemia"
is art recognized and refers to a progressive, malignant disease of the
blood-forming organs, marked by distorted proliferation and development
of leukocytes and their precursors in the blood and bone marrow.

[0068]The term "modulate" refers to increases or decreases in the activity
of a cell in response to exposure to a compound of the invention, e.g.,
the inhibition of proliferation and/or induction of differentiation of at
least a sub-population of cells in an animal such that a desired end
result is achieved, e.g., a therapeutic result. In preferred embodiments,
this phrase is intended to include hyperactive conditions that result in
pathological disorders.

[0069]The common medical meaning of the term "neoplasia" refers to "new
cell growth" that results as a loss of responsiveness to normal growth
controls, e.g. to neoplastic cell growth. A "hyperplasia" refers to cells
undergoing an abnormally high rate of growth. However, as used herein,
the terms neoplasia and hyperplasia can be used interchangably, as their
context will reveal, referring to generally to cells experiencing
abnormal cell growth rates. Neoplasias and hyperplasias include "tumors,"
which may be either benign, premalignant or malignant.

[0073]The terms "polycyclyl" or "polycyclic radical" refer to the radical
of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons
are common to two adjoining rings, e.g., the rings are "fused rings".
Rings that are joined through non-adjacent atoms are termed "bridged"
rings. Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,
amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl,
alkylaryl, or an aromatic or heteroaromatic moiety.

[0074]The term "prodrug" includes compounds with moieties which can be
metabolized in vivo. Generally, the prodrugs are metabolized in vivo by
esterases or by other mechanisms to active drugs. Examples of prodrugs
and their uses are well known in the art (See, e.g., Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). The prodrugs can be
prepared in situ during the final isolation and purification of the
compounds, or by separately reacting the purified compound in its free
acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups
can be converted into esters via treatment with a carboxylic acid.
Examples of prodrug moieties include substituted and unsubstituted,
branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid
esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters
(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,
acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,
pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl
esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or
methoxy substituents) aryl and aryl-lower alkyl esters, amides,
lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred
prodrug moieties are propionoic acid esters and acyl esters. Prodrugs
which are converted to active forms through other mechanisms in vivo are
also included.

[0075]The language "a prophylactically effective anti-neoplastic amount"
of a compound refers to an amount of a vitamin D3 compound of the
formula (I) or otherwise described herein which is effective, upon single
or multiple dose administration to the patient, in preventing or delaying
the occurrence of the onset of a neoplastic disease state.

[0076]The term "psoriasis" is intended to have its medical meaning,
namely, a disease which afflicts primarily the skin and produces raised,
thickened, scaling, nonscarring lesions. The lesions are usually sharply
demarcated erythematous papules covered with overlapping shiny scales.
The scales are typically silvery or slightly opalescent. Involvement of
the nails frequently occurs resulting in pitting, separation of the nail,
thickening and discoloration. Psoriasis is sometimes associated with
arthritis, and it may be crippling.

[0077]The language "reduced toxicity" is intended to include a reduction
in any undesired side effect elicited by a vitamin D3 compound when
administered in vivo, e.g., a reduction in the hypercalcemic activity.

[0078]The term "sarcoma" is art recognized and refers to malignant tumors
of mesenchymal derivation.

[0079]The term "secosteroid" is art-recognized and includes compounds in
which one of the cyclopentanoperhydro-phenanthrene rings of the steroid
ring structure is broken. 1α, 25(OH)2D3 and analogs
thereof are hormonally active secosteroids. In the case of vitamin
D3, the 9-10 carbon-carbon bond of the B-ring is broken, generating
a seco-B-steroid. The official IUPAC name for vitamin D3 is
9,10-secocholesta-5,7,10(19)-trien-3B-ol. For convenience, a 6-s-trans
conformer of 1α, 25(OH)2D3 is illustrated herein having
all carbon atoms numbered using standard steroid notation.

In the formulas presented herein, the various substituents on ring A are
illustrated as joined to the steroid nucleus by one of these notations: a
dotted line or indicating a substituent which is in the
α-orientation (i.e., above the plane of the ring), a wedged solid
line () indicating a substituent which is in the α-orientation
(i.e., below the plane of the molecule), or a wavy line indicating that
a substituent may be either above or below the plane of the ring. In
regard to ring A, it should be understood that the stereochemical
convention in the vitamin D field is opposite from the general chemical
field, wherein a dotted line indicates a substituent on Ring A which is
in an α-orientation (i.e., below the plane of the molecule), and a
wedged solid line indicates a substituent on ring A which is in the
β-orientation (i.e., above the plane of the ring). As shown, the A
ring of the hormone 1α, 25(OH)2D3 contains two asymmetric
centers at carbons 1 and 3, each one containing a hydroxyl group in
well-characterized configurations, namely the 1α- and
3β-hydroxyl groups. In other words, carbons 1 and 3 of the A ring
are said to be "chiral carbons" or "carbon centers."

[0080]Furthermore the indication of stereochemistry across a carbon-carbon
double bond is also opposite from the general chemical field in that "Z"
refers to what is often referred to as a "cis" (same side) conformation
whereas "E" refers to what is often referred to as a "trans" (opposite
side) conformation. As shown, the A ring of the hormone 1-alpha,
25(OH)2D3 contains two asymmetric centers at carbons 1 and 3,
each one containing a hydroxyl group in well-characterized
configurations, namely the 1-alpha- and 3-beta-hydroxyl groups. In otlher
words, carbons 1 and 3 of the A ring are said to be "chiral carbons" or
"chiral carbon centers." Regardless, both configurations, cis/trans
and/or Z/E are contemplated for the compounds for use in the present
invention.

[0081]With respect to the nomenclature of a chiral center, the terms "d"
and "l" configuration are as defined by the IUPAC Recommendations. As to
the use of the terms, diastereomer, racemate, epimer and enantiomer,
these will be used in their normal context to describe the
stereochemistry of preparations.

[0082]Also, throughout the patent literature, the A ring of a vitamin D
compound is often depicted in generic formulae as any one of the
following structures:

wherein X1 and X2 are defined as H or ═CH2; or

wherein X1 and X2 are defined as H2 or CH2.

[0083]Although there does not appear to be any set convention, it is clear
that one of ordinary skill in the art understands either formula I or II
to represent an A ring in which, for example, X1 is ═CH2
and X2 is defined as H2, as follows:

For purposes of the instant invention, formula II will be used in all
generic structures.

[0084]The term "sulfhydryl" or "thiol" means --SH.

[0085]The term "subject" includes organisms which are capable of suffering
from a vitamin D3 associated state or who could otherwise benefit
from the administration of a vitamin D3 compound of the invention,
such as human and non-human animals. Preferred human animals include
human patients suffering from or prone to suffering from a vitamin
D3 associated state, as described herein. The term "non-human
animals" of the invention includes all vertebrates, e.g., mammals, e.g.,
rodents, e.g., mice, and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.

[0086]The phrases "systemic administration," "administered systemically",
"peripheral administration" and "administered peripherally" as used
herein mean the administration of a vitamin D3 compound(s), drug or
other material, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example, subcutaneous
administration.

[0087]The language "therapeutically effective anti-neoplastic amount" of a
vitamin D3 compound of the invention refers to an amount of an agent
which is effective, upon single or multiple dose administration to the
patient, in inhibiting the growth of a neoplastic vitamin
D3-responsive cells, or in prolonging the survivability of the
patient with such neoplastic cells beyond that expected in the absence of
such treatment.

[0088]The language "transplant rejection" refers to an immune reaction
directed against a transplanted organ(s) from other human donors
(allografts) or from other species such as sheep, pigs, or non-human
primates (xenografts). Therefore, the method of the invention is useful
for preventing an immune reaction to transplanted organs from other human
donors (allografts) or from other species (xenografts). Such tissues for
transplantation include, but are not limited to, heart, liver, kidney,
lung, pancreas, pancreatic islets, bone marrow, brain tissue, cornea,
bone, intestine, skin, and hematopoietic cells. Also included within this
definition is "graft versus host disease" of "GVHD," which is a condition
where the graft cells mount an immune response against the host.
Therefore, the method of the invention is useful in preventing graft
versus host disease in cases of mismatched bone marrow or lymphoid tissue
transplanted for the treatment of acute leukemia, aplastic anemia, and
enzyme or immune deficiencies, for example. The term "transplant
rejection" also includes disease symptoms characterized by loss of organ
function. For example, kidney rejection would be characterized by a
rising creatine level in blood. Heart rejection is characterized by an
endomyocardial biopsy, while pancreas rejection is characterized by
rising blood glucose levels. Liver rejection is characterized by the
levels of transaminases of liver origin and bilirubin levels in blood.
Intestine rejection is determined by biopsy, while lung rejection is
determined by measurement of blood oxygenation.

[0089]The term Vitamin D. Receptor ("VDR") is intended to include members
of the type II class of steroid/thyroid superfamily of receptors
(Stunnenberg, H. G. (1993) Bio Essays 15(5):309-15), which are able to
bind and transactivate through the vitamin D response element (VDRE) in
the absence of a ligand (Damm et al. (1989) Nature 339:593-97; Sap et al.
Nature 343:177-180).

[0091]The language "vitamin D3 associated state" is a state which can
be prevented, treated or otherwise ameliorated by administration of one
or more compounds of the invention. Vitamin D3 associated states
include ILT3-associated disorders, disorders characterized by an aberrant
activity of a vitamin D3-responsive cell, disorders characterized by
a deregulation of calcium and phosphate metabolism, and other disorders
or states described herein.

[0092]The term "vitamin D3-responsive cell" includes any cell which
is capable of responding to a vitamin D3 compound having the formula
I or otherwise described herein, or is associated with disorders
involving an aberrant activity of hyperproliferative skin cells,
parathyroid cells, neoplastic cells, immune cells, and bone cells. These
cells can respond to vitamin D3 activation by triggering genomic
and/or non-genomic responses that ultimately result in the modulation of
cell proliferation, differentiation survival, and/or other cellular
activities such as hormone secretion. In a preferred embodiment, the
ultimate responses of a cell are inhibition of cell proliferation and/or
induction of differentiation-specific genes. Exemplary vitamin D3
responsive cells include immune cells, bone cells, neuronal cells,
endocrine cells, neoplastic cells, epidermal cells, endodermal cells,
smooth muscle cells, among others.

[0093]With respect to the nomenclature of a chiral center, terms "d" and
"I" configuration are as defined by the IUPAC Recommendations. As to the
use of the terms, diastereomer, racemate, epimer and enantiomer will be
used in their normal context to describe the stereochemistry of
preparations.

2. Vitamin D3 Compounds of the Invention

[0094]Prominent features of the vitamin D3 compounds of the invention
included 1,3-dihydroxy substitution in the A ring, a 20-cyclopropyl group
in the side chain, and a 16-ene double bond in the B ring. U.S. Pat. No.
6,492,353B1 to Manchand et al. describes 1,3-dihydroxy, 20-cyclopropyl
vitamin D3 compounds. However, any such compounds specifically
disclosed in U.S. Pat. No. 6,492,353B1 are excluded from the appended
claims.

[0095]The vitamin D3 compounds of formula I above exert a full
spectrum of 1,25(OH)2D3 biological activities such as binding
to the specific nuclear receptor VDR, suppression of the increased
parathyroid hormone levels in 5,6-nephrectomized rats, suppression of
INF-γ release in MLR cells, stimulation of HL-60 leukemia cell
differentiation and inhibition of solid tumor cell proliferation. It is
well known that in vivo and in cellular cultures 1,25-(OH)2D3
undergoes a cascade of metabolic modifications initiated by the influence
of 24R-hydroxylase enzyme. First 24R-hydroxy metabolite is formed, which
is oxidized to 24-keto intermediate, and then 23S-hydroxylation and
fragmentation produce the fully inactive calcitroic acid.

[0096]Thus, in one aspect, the invention provides a vitamin D3
compound of formula I:

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2;R1 is hydroxyl or halogen;R2 and R3 taken
together with C20 form C3-C6 cycloalkyl;R4 and
R5 are each independently alkyl, or haloalkyl;R6 is hydrogen,
C1-C4 alkyl, hydroxyalkyl, or haloalkyl, with the understanding
that R6 is absent when B is a triple bond; andpharmaceutically
acceptable esters, salts, and prodrugs thereof.

[0097]In one embodiment, R1 is hydroxyl. In another embodiment, B is
a single, double, or triple bond. In another embodiment, X1 is
CH2 and X2 is H2, or are each H2. In a further
embodiment, R4 and R5 are each independently alkyl or
haloalkyl, preferably alkyl or trihaloalkyl, preferably, methyl or
trifluoromethyl. In another embodiment, R2 and R3 taken
together with C20 form C3-C6 cycloalkyl, preferably
cyclopropyl.

[0098]In another embodiment, the invention provides a vitamin D3
compound of formula I-a

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2; andR4 and R5 are each independently alkyl or
haloalkyl.

[0099]In a further embodiment, X1 is CH2 and X2 is H2.
In a preferred embodiment, B is a triple bond, and R4 and R5
are alkyl or haloalkyl. Preferably, R4 and R5 are preferably
alkyl or trihaloalkyl, preferably methyl or trifluoromethyl. In another
embodiment, B is a double bond and R4 and R5 are haloalkyl,
preferably trihaloalkyl, preferably trifluoromethyl. In yet another
preferred embodiment, B is a single bond and R4 and R5 are
alkyl, preferably methyl.

[0100]In another embodiment, X1 and X2 are each H2. In a
preferred embodiment, B is a triple bond and R4 and R5 are
alkyl or haloalkyl. Preferably, R4 and R5 are alkyl or
trihaloalkyl, preferably methyl or trifluoromethyl. In another preferred
embodiment, B is a double bond and R4 and R5 are haloalkyl,
preferably trihaloalkyl, preferably trifluoromethyl. In yet another
embodiment, B is a single bond and R4 and R5 are alkyl,
preferably methyl.

[0105]The structures of some of the compounds of the invention include
asymmetric carbon atoms. Accordingly, the isomers arising from such
asymmetry (e.g., all enantiomers and diastereomers) are included within
the scope of the invention, unless indicated otherwise. Such isomers can
be obtained in substantially pure form by classical separation techniques
and/or by stereochemically controlled synthesis.

[0106]Naturally occurring or synthetic isomers can be separated in several
ways known in the art. Methods for separating a racemic mixture of two
enantiomers include chromatography using a chiral stationary phase (see,
e.g., "Chiral Liquid Chromatography," W. J. Lough, Ed. Chapman and Hall,
New York (1989)). Enantiomers can also be separated by classical
resolution techniques. For example, formation of diastereomeric salts and
fractional crystallization can be used to separate enantiomers. For the
separation of enantiomers of carboxylic acids, the diastereomeric salts
can be formed by addition of enantiomerically pure chiral bases such as
brucine, quinine, ephedrine, strychnine, and the like. Alternatively,
diastereomeric esters can be formed with enantiomerically pure chiral
alcohols such as menthol, followed by separation of the diastereomeric
esters and hydrolysis to yield the free, enantiomerically enriched
carboxylic acid. For separation of the optical isomers of amino
compounds, addition of chiral carboxylic or sulfonic acids, such as
camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can
result in formation of the diastereomeric salts.

3. Uses of the Vitamin D3 Compounds of the Invention

[0107]In one aspect, the invention provides a method for treating a
subject for a vitamin D3 associated state, comprising administering
to said subject in need thereof an effective amount of a vitamin D3
compound of, of formula I the invention, including compounds of formulas
Ia and Ib, and the preferred compounds herein above described, such that
said subject is treated for said vitamin D3 associated state.

[0108]In one embodiment, the method, further comprises the step of
obtaining the vitamin D3 compound. In another embodiment, the method
further comprising identifying a subject in need of treatment for a
vitamin D3 associated state.

[0109]In one embodiment, the vitamin D3 associated state is an
ILT3-associated disorder. In a further embodiment, the ILT3-associated
disorder is an immune disorder.

[0110]In another embodiment, the immune disorder is an autoimmune
disorder.

[0112]In another embodiment, the immune disorder is transplant rejection.

[0113]In another embodiment, the autoimmune disorder is type I insulin
dependent diabetes mellitus.

[0114]In yet another embodiment, the vitamin D3 associated state is a
disorder characterized by an aberrant activity of a vitamin
D3-responsive cell. In another embodiment, the disorder comprises an
aberrant activity of a hyperproliferative skin cell. In yet another
embodiment, the disorder is selected from psoriasis, basal cell carcinoma
and keratosis.

[0115]In another embodiment, the disorder is psoriasis. In a further
embodiment, the Vitamin D3 compound used to treat psoriasis has the
formula I-a

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2; andR4 and R5 are each independently alkyl, or
haloalkyl.

[0116]In a further embodiment, vitamin D3 compound is
1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

[0117]In another embodiment, the disorder comprises an aberrant activity
of an endocrine cell. In a further embodiment, the endocrine cell is a
parathyroid cell and the aberrant activity is processing and/or secretion
of parathyroid hormone.

[0118]In yet another embodiment, the disorder is secondary
hyperparathyroidism.

[0119]In still another embodiment, the disorder comprises an aberrant
activity of a bone cell. In a further embodiment, disorder is selected
from osteoporosis, osteodystrophy, senile osteoporosis, osteomalacia,
rickets, osteitis fibrosa cystica, and renal osteodystrophy. In one
embodiment, the disorder is osteoporosis. In another embodiment, the
vitamin D3 compound used to treat osteoporosis has the formula I-a

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2; andR4 and R5 are each independently alkyl, or
haloalkyl.

[0120]In a further embodiment, the vitamin D3 compound used to treat
osteoporosis is
1,25-Dihydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-19-nor-chole-
calciferol:

[0121]In a further embodiment, the vitamin D3 compound used to treat
osteoporosis is 1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

[0122]In another embodiment, the disorder is cirrhosis or chronic renal
disease.

[0123]In another embodiment, the disorder is hypertension.

[0124]In another embodiment, the compound of the invention suppresses
expression of renin, thereby treating the subject for hypertension. In a
further embodiment, the Vitamin D3 compound used to suppress rennin
expression has the formula I-a

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2; andR4 and R5 are each independently alkyl, or
haloalkyl.

[0125]In another embodiment, the vitamin D3 compound used to suppress
rennin expression has the formula I-b

wherein:B is single, double, or triple bond;X1 and X2 are each
independently H2 or CH2, provided X1 and X2 are not
both CH2; andR4 and R5 are each independently alkyl, or
haloalkyl.In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1,25-Dihydroxy-16-ene-23-yne-20-cyclopyl-cholecalciferol:

[0126]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1,25-Dihydroxy-16-ene-23-yne-20-cyclopropyl-19-nor-cholecalciferol:

[0127]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1,25-Dihydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-cholecalcifer-
ol:

[0128]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1,25-Dihydroxy-16-ene-20-cyclopropyl-19-nor-cholecalciferol:

[0129]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1,25-Dihydroxy-16-ene-20-cyclopropyl-cholecalciferol:

[0130]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1α-Fluoro-25-hydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-c-
holecalciferol:

[0131]In a further embodiment, the vitamin D3 compound used to
suppress rennin expression is
1α-Fluoro-25-hydroxy-16,23Z-diene-20-cyclopropyl-26,27-hexafluoro-c-
holecalciferol:

[0132]In another embodiment, the disorder is benign prostate hypertrophy.

[0133]In another embodiment, the disorder is neoplastic disease. In a
further embodiment, the neoplastic disease is selected from the group
consisting of leukemia, lymphoma, melanoma, osteosarcoma, colon cancer,
rectal cancer, prostate cancer, bladder cancer, and malignant tumors of
the lung, breast, gastrointestinal tract, and genitourinary tract. In
another embodiment, the neoplastic disease is bladder cancer.

[0134]In another embodiment, the disorder is neuronal loss. In a further
embodiment, the disorder is selected from the group consisting of
Alzheimer's Disease, Pick's Disease, Parkinson's Disease, Vascular
Disease, Huntington's Disease, and Age-Associated Memory Impairment.

[0135]In another embodiment, the disorder is uveitis.

[0136]In another embodiment, the disorder is interstitial cystitis.

[0137]In another embodiment, the disorder is characterized by an aberrant
activity of a vitamin D3-responsive smooth muscle cell. In one
embodiment, the disorder is uterine myomas. In another embodiment, the
disorder is hyperproliferative vascular disease selected from the group
consisting of hypertension-induced vascular remodeling, vascular
restenosis, and atherosclerosis. In yet another a further embodiment, the
disorder is arterial hypertension.

[0138]In one embodiment, the invention provides a method of ameliorating a
deregulation of calcium and phosphate metabolism, comprising
administering to a subject a therapeutically effective amount of a
compound of the invention, so as to ameliorate the deregulation of the
calcium and phosphate metabolism. In a further embodiment, the
deregulation of the calcium and phosphate metabolism leads to
osteoporosis.

[0139]In yet another embodiment, the invention provides a method of
modulating the expression of an immunoglobulin-like transcript 3 (ILT3)
surface molecule in a cell, comprising contacting said cell with a
compound of the invention in an amount effective to modulate the
expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule
in said cell. In another embodiment, the cell is within a subject.

[0140]In still another embodiment, the invention provides a method of
treating an ILT3-associated disorder in a subject, comprising
administering to said subject a compound of the invention, in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
treating said ILT3-associated disorder in said subject. In one
embodiment, the ILT3-associated disorder is an immune disorder. In
another embodiment, the immune disorder is an autoimmune disorder. In
another embodiment, the autoimmune disorder is type insulin dependent
diabetes mellitus.

[0141]In one embodiment, the invention provides a method of inducing
immunological tolerance in a subject, comprising administering to said
subject a compound of the invention, in an amount effective to modulate
the expression of an ILT3 surface molecule, thereby inducing
immunological tolerance in said subject. In one embodiment, the
immunological tolerance is induced in an antigen-presenting cell. In one
embodiment, the antigen-presenting cell is selected from the group
consisting of dendritic cells, monocytes, and macrophages.

[0142]In another embodiment, the invention provides a method of inhibiting
transplant rejection in a subject comprising administering to a subject a
compound of the invention, in an amount effective to modulate the
expression of an ILT3 surface molecule, thereby inhibiting transplant
rejection in said subject. In one embodiment, the transplant is a solid
organ transplant. In one embodiment, the transplant is a pancreatic islet
transplant. In one embodiment, the transplant is a bone marrow
transplant.

[0143]In another embodiment, the invention provides a method for
modulating immunosuppressive activity by an antigen-presenting cell,
comprising contacting an antigen-presenting cell with a compound of the
invention, in an amount effective to modulate ILT3 surface molecule
expression, thereby modulating said immunosuppressive activity by said
antigen-presenting cell.

[0144]In a further embodiment, the cell is an antigen-presenting cell. In
another embodiment, antigen-presenting cell is selected from the group
consisting of dendritic cells, monocytes, and macrophages.

[0145]In yet another embodiment, the invention provides a method for
preventing or treating bladder dysfunction in a subject in need thereof
by administering an effective amount of a compound of the invention,
thereby to prevent or treat bladder dysfunction in said subject.

[0146]In one embodiment, the bladder dysfunction is characterized by the
presence of bladder hypertrophy. In another embodiment, the bladder
dysfunction is overactive bladder. In another embodiment, the subject is
male. In another embodiment, the male is concurrently suffering from BPH.
In one embodiment, the subject is female.

[0147]In a further embodiment, the invention provides a method wherein the
vitamin D3 compound is administered in combination with a
pharmaceutically acceptable carrier.

[0148]In yet another embodiment, the invention provides a method wherein
said vitamin D3 compound is administered to the subject using a
pharmaceutically-acceptable formulation.

[0149]In still another embodiment, the invention provides a method wherein
said pharmaceutically-acceptable formulation provides sustained delivery
of said vitamin D3 compound to a subject for at least four weeks
after the pharmaceutically-acceptable formulation is administered to the
subject.

[0150]In one embodiment, the invention provides a method, wherein the
expression of said immunoglobulin-like transcript 3 (ILT3) surface
molecule is upregulated.

[0151]In another embodiment, the invention provides a method wherein the
compound is formulated in a pharmaceutical composition together with a
pharmaceutically acceptable diluent or carrier. In another embodiment,
the invention provides a method, wherein said compound is a Vitamin D
receptor agonist.

[0152]In another embodiment, the invention provides a method, wherein the
subject is a mammal, preferably a human.

[0153]In further embodiment, the compound is administered orally. In
another embodiment, the compound is administered intravenously. In
another embodiment, the compound is administered topically. In another
embodiment, the compound is administered parenterally.

[0154]In yet another embodiment, the compound is administered at a
concentration of 0.001 μg-100 μg/kg of body weight.

[0155]In another aspect, the invention provides a pharmaceutical
composition, comprising an effective amount of a compound of the
invention, and a pharmaceutically acceptable diluent or carrier. In one
embodiment, the effective amount is effective to treat a vitamin D3
associated state. In another embodiment, the invention provides a
pharmaceutical composition, wherein said vitamin D3 associated state
is an ILT3-associated disorder. In another embodiment, the invention
provides a pharmaceutical composition, wherein said vitamin D3
associated state is a disorder characterized by an aberrant activity of a
vitamin D3-responsive cell. In another embodiment, the invention
provides a pharmaceutical composition, wherein said vitamin D3
associated state is bladder dysfunction. In another embodiment, the
invention provides a pharmaceutical composition, wherein said disorder is
hypertension.

[0156]In one aspect, the invention provides a packaged formulation for use
in the treatment of a vitamin D3 associated state, comprising a
pharmaceutical composition comprising a compound of the invention, and
instructions for use in the treatment of a vitamin D3 associated
state. In one embodiment, the invention provides a package formulation
wherein said vitamin D3 associated state is an ILT3-associated
disorder. In another embodiment, the invention provides a packaged
formulation, wherein said vitamin D3 associated state is a disorder
characterized by an aberrant activity of a vitamin D3-responsive
cell. In another embodiment, the invention provides a packaged
formulation, wherein said vitamin D3 associated state is bladder
dysfunction.

[0157]In certain embodiments, the methods of the invention include
administering to a subject a therapeutically effective amount of a
vitamin D3 compound in combination with another pharmaceutically
active compound. Examples of pharmacuetically active compounds include
compounds known to treat autoimmune disorders, e.g., immunosuppressant
agents such as cyclosporin A, rapamycin, desoxyspergualine, FK 506,
steroids, azathioprine, anti-T cell antibodies and monoclonal antibodies
to T cell subpopulations. Other pharmaceutically active compounds that
may be used can be found in Harrison's Principles of Internal Medicine,
Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; and
the Physicians Desk Reference 50th Edition 1997, Oradell N.J., Medical
Economics Co., the complete contents of which are expressly incorporated
herein by reference. The vitamin D3 compound and the
pharmaceutically active compound may be administered to the subject in
the same pharmaceutical composition or in different pharmaceutical
compositions (at the same time or at different times).

[0158]A. Hyperproliferative Conditions

[0159]In another aspect, the present invention provides a method of
treating a subject for a disorder characterized by aberrant activity of a
vitamin D3-responsive cell. The method involves administering to the
subject an effective amount of a pharmaceutical composition of a vitamin
D3 compound of formula I or otherwise described herein such that the
activity of the cell is modulated.

[0160]In certain embodiments, the cells to be treated are
hyperproliferative cells. As described in greater detail below, the
vitamin D3 compounds of the invention can be used to inhibit the
proliferation of a variety of hyperplastic and neoplastic tissues. In
accordance with the present invention, vitamin D3 compounds of the
invention can be used in the treatment of both pathologic and
non-pathologic proliferative conditions characterized by unwanted growth
of vitamin D3-responsive cells, e.g., hyperproliferative skin cells,
immune cells, and tissue having transformed cells, e.g., such as
carcinomas, sarcomas and leukemias. In other embodiments, the cells to be
treated are aberrant secretory cells, e.g., parathyroid cells, immune
cells.

[0161]The use of vitamin D compounds in treating hyperproliferative
conditions has been limited because of their hypercalcemic effects. Thus,
vitamin D3 compounds of the invention can provide a less toxic
alternative to current methods of treatment.

[0162]In one embodiment, the invention features a method for inhibiting
the proliferation and/or inducing the differentiation of a
hyperproliferative skin cell, e.g., an epidermal or an epithelial cell,
e.g., a keratinocytes, by contacting the cells with a vitamin D3
compound of the invention. In general, the method includes a step of
contacting a pathological or non-pathological hyperproliferative cell
with an effective amount of such vitamin D3 compound to promote the
differentiation of the hyperproliferative cells The present method can be
performed on cells in culture, e.g., in vitro or ex vivo, or can be
performed on cells present in an animal subject, e.g., as part of an in
vivo therapeutic protocol. The therapeutic regimen can be carried out on
a human or any other animal subject.

[0164]In an illustrative example, vitamin D3 compounds of the
invention can be used to inhibit the hyperproliferation of keratinocytes
in treating diseases such as psoriasis by administering an effective
amount of these compounds to a subject in need of treatment. The term
"psoriasis" is intended to have its medical meaning, namely, a disease
which afflicts primarily the skin and produces raised, thickened,
scaling, nonscarring lesions. The lesions are usually sharply demarcated
erythematous papules covered with overlapping shiny scales. The scales
are typically silvery or slightly opalescent. Involvement of the nails
frequently occurs resulting in pitting, separation of the nail,
thickening and discoloration. Psoriasis is sometimes associated with
arthritis, and it may be crippling. Hyperproliferation of keratinocytes
is a key feature of psoriatic epidermal hyperplasia along with epidermal
inflammation and reduced differentiation of keratinocytes. Multiple
mechanisms have been invoked to explain the keratinocyte
hyperproliferation that characterizes psoriasis. Disordered cellular
immunity has also been implicated in the pathogenesis of psoriasis.

[0165]B. Neoplasia

[0166]The invention also features methods for inhibiting the proliferation
and/or reversing the transformed phenotype of vitamin D3-responsive
hyperproliferative cells by contacting the cells with a vitamin D3
compound of formula I or otherwise described herein. In general, the
method includes a step of contacting pathological or non-pathological
hyperproliferative cells with an effective amount of a vitamin D3
compound of the invention for promoting the differentiation of the
hyperproliferative cells. The present method can be performed on cells in
culture, e.g., in vitro or ex vivo, or can be performed on cells present
in an animal subject, e.g., as part of an in vivo therapeutic protocol.
The therapeutic regimen can be carried out on a human or other subject.

[0169]In certain embodiments, the vitamin D3 compounds of the
invention can be used in combinatorial therapy with conventional cancer
chemotherapeutics. Conventional treatment regimens for leukemia and for
other tumors include radiation, drugs, or a combination of both. In
addition to radiation, the following drugs, usually in combinations with
each other, are often used to treat acute leukemias: vincristine,
prednisone, methotrexate, mercaptopurine, cyclophosphamide, and
cytarabine. In chronic leukemia, for example, busulfan, melphalan, and
chlorambucil can be used in combination. All of the conventional
anti-cancer drugs are highly toxic and tend to make patients quite ill
while undergoing treatment. Vigorous therapy is based on the premise that
unless every leukemic cell is destroyed, the residual cells will multiply
and cause a relapse.

[0170]The subject method can also be useful in treating malignancies of
the various organ systems, such as affecting lung, breast, lymphoid,
gastrointestinal, and genitourinary tract as well as adenocarcinomas
which include malignancies such as most colon cancers, renal-cell
carcinoma, prostate cancer and/or testicular tumors, non-small cell
carcinoma of the lung, cancer of the small intestine, cancer of the
esophagus, and bladder cancer.

[0172]Determination of a therapeutically effective anti-neoplastic amount
or a prophylactically effective anti-neoplastic amount of the vitamin
D3 compound of the invention, can be readily made by the physician
or veterinarian (the "attending clinician"), as one skilled in the art,
by the use of known techniques and by observing results obtained under
analogous circumstances. The dosages may be varied depending upon the
requirements of the patient in the judgment of the attending clinician,
the severity of the condition being treated and the particular compound
being employed. In determining the therapeutically effective
antineoplastic amount or dose, and the prophylactically effective
antineoplastic amount or dose, a number of factors are considered by the
attending clinician, including, but not limited to: the specific
hyperplastic/neoplastic cell involved; pharmacodynamic characteristics of
the particular agent and its mode and route of administration; the
desirder time course of treatment; the species of mammal; its size, age,
and general health; the specific disease involved; the degree of or
involvement or the severity of the disease; the response of the
individual patient; the particular compound administered; the mode of
administration; the bioavailability characteristics of the preparation
administered; the dose regimen selected; the kind of concurrent treatment
(i.e., the interaction of the vitamin D3 compounds of the invention
with other co-administered therapeutics); and other relevant
circumstances. U.S. Pat. No. 5,427,916, for example, describes method for
predicting the effectiveness of antineoplastic therapy in individual
patients, and illustrates certain methods which can be used in
conjunction with the treatment protocols of the instant invention.

[0173]Treatment can be initiated with smaller dosages which are less than
the optimum dose of the compound. Thereafter, the dosage should be
increased by small increments until the optimum effect under the
circumstances is reached. For convenience, the total daily dosage may be
divided and administered in portions during the day if desired. A
therapeutically effective antineoplastic amount and a prophylactically
effective anti-neoplastic amount of a vitamin D3 compound of the
invention is expected to vary from about 0.1 milligram per kilogram of
body weight per day (mg/kg/day) to about 100 mg/kg/day.

[0174]Compounds which are determined to be effective for the prevention or
treatment of tumors in animals, e.g., dogs, rodents, may also be useful
in treatment of tumors in humans. Those skilled in the art of treating
tumors in humans will know, based upon the data obtained in animal
studies, the dosage and route of administration of the compound to
humans. In general, the dosage and route of administration in humans is
expected to be similar to that in animals.

[0175]The identification of those patients who are in need of prophylactic
treatment for hyperplastic/neoplastic disease states is well within the
ability and knowledge of one skilled in the art. Certain of the methods
for identification of patients which are at risk of developing neoplastic
disease states which can be treated by the subject method are appreciated
in the medical arts, such as family history of the development of a
particular disease state and the presence of risk factors associated with
the development of that disease state in the subject patient. A clinician
skilled in the art can readily identify such candidate patients, by the
use of, for example, clinical tests, physical examination and
medical/family history.

[0176]C. Immuniological Activity

[0177]Healthy individuals protect themselves against foreign invaders
using many different mechanisms, including physical barriers, phagocytic
cells in the blood and tissues, a class of immune cells known as
lymphocytes, and various blood-born molecules. All of these mechanisms
participate in defending individuals from a potentially hostile
environment. Some of these defense mechanisms, known as natural or innate
immunity, are present in an individual prior to exposure to infectious
microbes or other foreign macromolecules, are not enhanced by such
exposures, and do not discriminate among most foreign substances. Other
defense mechanisms, known as acquired or specific immunity, are induced
or stimulated by exposure of foreign substances, are exquisitely specific
for distinct macromolecules, and increase in magnitude and defensive
capabilities with each successive exposure to a particular macromolecule.
Substances that induce a specific immune response are known as antigens
(see, e.g., Abbas, A. et al., Cellular and Molecular Immunology, W.B.
Saunders Company, Philadelphia, 1991; Silverstein, A. M. A history of
Immunology, San Diego, Academic Press, 1989; Unanue A. et al., Textbook
of Immunology, 2nd ed. Williams and Wilkens, Baltimore, 1984).

[0178]One of the most remarkable properties of the immune system is its
ability to distinguish between foreign antigens and self-antigens.
Therefore, the lymphocytes in each individual are able to recognize and
respond to many foreign antigens but are normally unresponsive to the
potentially antigenic substances present in the individual. This
immunological unresponsiveness is referred to as immune tolerance (see,
e.g., Burt R K et al. (2002) Blood 99:768; Coutinho, A. et al. (2001)
Immunol. Rev. 182:89; Schwartz, R H (1990) Science 248:1349; Miller, J.
F. et al. (1989) Immunology Today 10:53).

[0179]Self-tolerance is an acquired process that has to be learned by the
lymphocytes of each individual. It occurs in part because lymphocytes
pass through a stage in their development when an encounter with antigen
presented by antigen-presenting cells (APCs) leads to their death or
inactivation in a process known as positive and negative selection (see,
e.g., Debatin K M (2001) Ann. Hematol. 80 Suppl 3:B29; Abbas, A. (1991),
supra). Thus, potentially self-recognizing lymphocytes come into contact
with self-antigens at this stage of functional immaturity and are
prevented from developing to a stage at which they would be able to
respond to self-antigens. Autoimmunity arises when abnormalities in the
induction or maintenance of self-tolerance occur that result in a loss of
tolerance to a particular antigen(s) and a subsequent attack by the
host's immune system on the host's tissues that express the antigen(s)
(see, e.g., Boyton R J et al. (2002) Clin. Exp. Immunol. 127:4; Hagiwara
E. (2001) Ryumachi 41:888; Burt R K et al (2992) Blood 99:768).

[0180]The ability of the immune system to distinguish between self and
foreign antigens also plays a critical role in tissue transplantation.
The success of a transplant depends on preventing the immune system of
the host recipient from recognizing the transplant as foreign and, in
some cases, preventing the graft from recognizing the host tissues as
foreign. For example, when a host receives a bone marrow transplant, the
transplanted bone marrow may recognize the new host as foreign, resulting
in graft versus host disease (GVHD). Consequently, the survival of the
host depends on preventing both the rejection of the donor marrow as well
as rejection of the host by the graft immune reaction (see, e.g.,
Waldmann H et al. (2001) Int. Arch. Allergy Immunol. 126:11).

[0181]Currently, deleterious immune reactions that result in autoimmune
diseases and transplant rejections are prevented or treated using agents
such as steroids, azathioprine, anti-T cell antibodies, and more
recently, monoclonal antibodies to T cell subpopulations.
Immunosuppressive drugs such as cyclosporin A (CsA), rapamycin,
desoxyspergualine and FK-506 are also widely used.

[0182]Nonspecific immune suppression agents, such as steroids and
antibodies to lymphocytes, put the host at increased risk for
opportunisitc infection and development of tumors. Moreover, many
immunosuppressive drugs result in bone demineralization within the host
(see, e.g., Chhajed P N et al. (2002) Indian J. Chest Dis. Allied 44:31;
Wijdicks E F (2001) Liver Transpl. 7:937; Karamehic J et al. (2001) Med.
Arh. 55:243; U.S. Pat. No. 5,597,563 issued to Beschorner, W E and U.S.
Pat. No. 6,071,897 issued to DeLuca H F et al.). Because of the major
drawbacks associated with existing immunosuppressive modalities, there is
a need for a new approach for treating immune disorders, e.g., for
inducing immune tolerance in a host.

[0183]Thus, in another aspect, the invention provides a method for
modulating the activity of an immune cell by contacting the cell with a
vitamin D3 compound of formula I or otherwise described herein.

[0184]In one embodiment, the present invention provides a method for
suppressing immune activity in an immune cell by contacting a
pathological or non-pathological immune cell with an effective amount of
a vitamin D3 compound of the invention to thereby inhibit an immune
response relative to the cell in the absence of the treatment. The
present method can be performed on cells in culture, e.g., in vitro or ex
vivo, or can be performed on cells present in an animal subject, e.g., as
part of an in vivo therapeutic protocol. In vivo treatment can be carried
out on a human or other animal subject.

[0186]After identifying certain test compounds as effective suppresors of
an immune response in vitro, these compounds can be used in vivo as part
of a therapeutic protocol. Accordingly, another aspect of the invention
provides a method of suppressing an immune response, comprising
administering to a subject a pharmaceutical preparation of a vitamin
D3 compounds of the invention, so as to inhibit immune reactions
such as graft rejection, autoimmune disorders and inflammation.

[0187]In one embodiment, the invention provides a method for treating a
subject for a vitamin D3 associated state, wherein the vitamin
D3 associated state is an ILT3-associated disorder, by administering
to the subject an effective amount of a vitamin D3 compound of the
invention. In one embodiment, the ILT3-associated state is an immune
disorder. In certain embodiments, the immune disorder is an autoimmune
disorder. In a specific embodiment, the immune disorder is Type 1
diabetes mellitus. In other embodiments, the immune disorder is
transplant rejection.

[0189]Another aspect of the invention provides a method of modulating the
expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule
in a cell. The method includes contacting the cell with a compound of
formula I in an amount effective to modulate the expression of an
immunoglobulin-like transcript 3 (ILT3) surface molecule in the cell. In
one embodiment, cell is within a subject a subject. In another embodiment
the modulation is upregulation of expression. In other embodiment, the
modulation is downregulation of expression.

[0190]A related aspect of the invention provides a method of treating an
ILT3-associated disorder in a subject. The method includes administering
to the subject a compound of formula I in an amount effective to modulate
the expression of an ILT3 surface molecule, thereby treating the
ILT3-associated disorder in the subject.

[0191]In certain embodiments, the present invention provides methods and
compositions for treating immune disorders, such as, for example,
autoimmune disorders and transplant rejections, such as graft versus host
disease (GVHD). These embodiments of the invention are based on the
discovery that vitamin D3 compounds of the invention are able to
modulate the expression of immunoglobulin-like transcript 3 (ILT3) on
cells, e.g., antigen-presenting cells.

[0192]Accordingly, another aspect of the invention provides a method for
inhibiting transplant rejection in a subject. The method includes
administering to the subject a compound of formula I in an amount
effective to modulate the expression of an ILT3 surface molecule, thereby
inhibiting transplant rejection in the subject. In one embodiment, the
transplant is an organ transplant. In another embodiment, the transplant
is a pancreatic islet transplant. In yet another embodiment, the
transplant is a bone marrow transplant.

[0193]As described before, determination of a therapeutically effective
immunosuppressive amount can be readily made by the attending clinician,
as one skilled in the art, by the use of known techniques and by
observing results obtained under analogous circumstances. Compounds which
are determined to be effective in animals, e.g., dogs, rodents, may be
extrapolated accordingly to humans by those skilled in the art. Starting
dose/regimen used in animals can be estimated based on prior studies. For
example, doses of vitamin D3 compounds of the invention to treat
autoimmune disorders in rodents can be initially estimated in the range
of 0.1 g/kg/day to 1 g/kg/day, administered orally or by injection.

[0194]Those skilled in the art will know based upon the data obtained in
animal studies, the dosage and route of administration in humans is
expected to be similar to that in animals. Exemplary dose ranges to be
used in humans are from 0.25 to 10 μg/day, preferably 0.5 to 5
μg/day per adult (U.S. Pat. No. 4,341,774).

D. Calcium and Phosphate Homeostasis

[0195]The present invention also relates to a method of treating in a
subject a disorder characterized by deregulation of calcium metabolism.
This method comprises contacting a pathological or non-pathological
vitamin D3 responsive cell with an effective amount of a vitamin
D3 compound of the invention to thereby directly or indirectly
modulate calcium and phosphate homeostasis. Techniques for detecting
calcium fluctuation in vivo or in vitro are known in the art.

[0196]Exemplary Ca++ homeostasis related assays include assays that
focus on the intestine where intestinal 45Ca2+ absorption is
determined either 1) in vivo (Hibberd K. A. and Norman A. W. (1969)
Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr.
91:319-323; Bickle D. D. et al. (1984) Endocrinology 114:260-267), or 2)
in vitro with everted duodenal sacs (Schachter D. et al. (1961) Am. J.
Physiol 200:1263-1271), or 3) on the genomic induction of
calbindin-D28k in the chick or of calbindin-D9k in the rat
(Thomasset M. et al. (1981) FEBS Lett. 127:13-16; Brehier A. and
Thomasset M. (1990) Endocrinology 127:580-587). The bone-oriented assays
include: 1) assessment of bone resorption as determined via the release
of Ca2+ from bone in vivo (in animals fed a zero Ca2+ diet)
(Hibberd K. A. and Norman A. W. (1969) Biochem. Pharmacol. 18:2347-2355;
Hurwitz S. et al. (1967) J. Nutr. 91:319-323), or from bone explants in
vitro (Bouillon R. et al. (1992) J. Biol. Chem. 267:3044-3051), 2)
measurement of serum osteocalcin levels [osteocalcin is an
osteoblast-specific protein that after its synthesis is largely
incorporated into the bone matrix, but partially released into the
circulation (or tissue culture medium) and thus represents a good market
of bone formation or turnover] (Bouillon R. et al. (1992) Clin. Chem.
38:2055-2060), or 3) bone ash content (Norman A. W. and Wong R. G. (1972)
J. Nutr. 102:1709-1718). Only one kidney-oriented assay has been
employed. In this assay, urinary Ca2+ excretion is determined
(Hartenbower D. L. et al. (1977) Walter de Gruyter, Berlin pp 587-589);
this assay is dependent upon elevations in the serum Ca2+ level and
may reflect bone Ca2+ mobilizing activity more than renal effects.
Finally, there is a "soft tissue calcification" assay that can be used to
detect the consequences of administration of a compound of the invention.
In this assay a rat is administered an intraperitoneal dose of
45Ca2+, followed by seven daily relative high doses of a
compound of the invention; in the event of onset of a severe
hypercalcemia, soft tissue calcification can be assessed by determination
of the 45Ca2+ level. In all these assays, vitamin D3
compounds of the invention are administered to vitamin D-sufficient or
-deficient animals, as a single dose or chronically (depending upon the
assay protocol), at an appropriate time interval before the end point of
the assay is quantified.

[0197]In certain embodiments, vitamin D3 compounds of the invention
can be used to modulate bone metabolism. The language "bone metabolism"
is intended to include direct or indirect effects in the formation or
degeneration of bone structures, e.g., bone formation, bone resorption,
etc., which may ultimately affect the concentrations in serum of calcium
and phosphate. This term is also intended to include effects of vitamin
D3 compounds in bone cells, e.g. osteoclasts and osteoblasts, that
may in turn result in bone formation and degeneration. For example, it is
known in the art, that vitamin D3 compounds exert effects on the
bone forming cells, the osteoblasts through genomic and non-genomic
pathways (Walters M. R. et al. (1982) J. Biol. Chem. 257:7481-7484;
Jurutka P. W. et al. (1993) Biochemistry 32:8184-8192; Mellon W. S. and
DeLuca H. F. (1980) J. Biol. Chem. 255:4081-4086). Similarly, vitamin
D3 compounds are known in the art to support different activities of
bone resorbing osteoclasts such as the stimulation of differentiation of
monocytes and mononuclear phagocytes into osteoclasts (Abe E. et al.
(1988) J. Bone Miner Res. 3:635-645; Takahashi N. et al. (1988)
Endocrinology 123:1504-1510; UdagawaN. et al. (1990) Proc. Natl. Acad.
Sci. USA 87:7260-7264). Accordingly, vitamin D3 compounds of the
invention that modulate the production of bone cells can influence bone
formation and degeneration.

[0198]The present invention provides a method for modulating bone cell
metabolism by contacting a pathological or a non-pathological bone cell
with an effective amount of a vitamin D3 compound of the invention
to thereby modulate bone formation and degeneration. The present method
can be performed on cells in culture, e.g., in vitro or ex vivo, or can
be performed in cells present in an animal subject, e.g., cells in vivo.
Exemplary culture systems that can be used include osteoblast cell lines,
e.g., ROS 17/2.8 cell line, monocytes, bone marrow culture system (Suda
T. et al (1990) Med. Res. Rev. 7:333-366; Suda T. et al. (1992) J. Cell
Biochem. 49:53-58) among others. Selected compounds can be further tested
in vivo, for example, animal models of osteopetrosis and in human disease
(Shapira F. (1993) Clin. Orthop. 294:34-44).

[0199]In a preferred embodiment, a method for treating osteoporosis is
provided, comprising administering to a subject a pharmaceutical
preparation of a vitamin D3 compound of the invention to thereby
ameliorate the condition relative to an untreated subject.

[0200]Vitamin D3 compounds of the invention can be tested in
ovarectomized animals, e.g., dogs, rodents, to assess the changes in bone
mass and bone formation rates in both normal and estrogen-deficient
animals. Clinical trials can be conducted in humans by attending
clinicians to determine therapeutically effective amounts of the vitamin
D3 compounds of the invention in preventing and treating
osteoporosis.

[0203]In yet another aspect, the present invention provides a method for
modulating hornone secretion of a vitamin D3-responsive cell, e.g.,
an endocrine cell. Hormone secretion includes both genomic and
non-genomic activities of vitamin D3 compounds of the invention that
control the transcription and processing responsible for secretion of a
given hormone e.g., parathyroid hormone (PTH), calcitonin, insulin,
prolactin (PRL) and TRH in a vitamin D3 responsive cell (Bouillon,
R. et al. (1995) Endocrine Reviews 16(2):235-237).

[0205]In certain embodiments, the vitamin D3 compounds of the present
invention can be used to inhibit parathyroid hormone (PTH) processing,
e.g., transcriptional, translational processing, and/or secretion of a
parathyroid cell as part of a therapeutic protocol. Therapeutic methods
using these compounds can be readily applied to all diseases, involving
direct or indirect effects of PTH activity, e.g., primary or secondary
responses.

[0206]Accordingly, therapeutic applications for the vitamin D3
compounds of the invention include treating diseases such as secondary
hyperparathyroidism of chronic renal failure (Slatopolsky E. et al.
(1990) Kidney Int. 38:S41-S47; Brown A. J. et al. (1989) J. Clin. Invest.
84:728-732). Determination of therapeutically affective amounts and dose
regimen can be performed by the skilled artisan using the data described
in the art.

[0207]F. Protection Against Neuronal Loss

[0208]In yet another aspect, the present invention provides a method of
protecting against neuronal loss by contacting a vitamin D3
responsive cell, e.g., a neuronal cell, with a vitamin D3 compound
of the invention to prevent or retard neuron loss. The language
"protecting against" is intended to include prevention, retardation,
and/or termination of deterioration, impairment, or death of a neurons.

[0209]Neuron loss can be the result of any condition of a neuron in which
its normal function is compromised. Neuron deterioration can be the
result of any condition which compromises neuron function which is likely
to lead to neuron loss. Neuron function can be compromised by, for
example, altered biochemistry, physiology, or anatomy of a neuron.
Deterioration of a neuron may include membrane, dendritic, or synaptic
changes which are detrimental to normal neuronal functioning. The cause
of the neuron deterioration, impairment, and/or death may be unknown.
Alternatively, it may be the result of age- and/or disease-related
changes which occur in the nervous system of a subject.

[0210]When neuron loss is described herein as "age-related", it is
intended to include neuron loss resulting from known and unknown bodily
changes of a subject which are associated with aging. When neuron loss is
described herein as "disease-related", it is intended to include neuron
loss resulting from known and unknown bodily changes of a subject which
are associated with disease. It should be understood, however, that these
terms are not mutually exclusive and that, in fact, many conditions that
result in the loss of neurons are both age- and disease-related.

[0211]Exemplary age-related diseases associated with neuron loss and
changes in neuronal morphology include, for example, Alzheimer's Disease,
Pick's Disease, Parkinson's Disease, Vascular Disease, Huntington's
Disease, and Age-Associated Memory Impairment. In Alzheimer's Disease
patients, neuron loss is most notable in the hippocampus, frontal,
parietal, and anterior temporal cortices, amygdala, and the olfactory
system. The most prominently affected zones of the hippocampus include
the CA1 region, the subiculum, and the entorhinal cortex. Memory loss is
considered the earliest and most representative cognitive change because
the hippocampus is well known to play a crucial role in memory. Pick's
Disease is characterized by severe neuronal degeneration in the neocortex
of the frontal and anterior temporal lobes which is sometimes accompanied
by death of neurons in the striatum. Parkinson's Disease can be
identified by the loss of neurons in the substantia nigra and the locus
ceruleus. Huntington's Disease is characterized by degeneration of the
intrastriatal and cortical cholinergic neurons and GABA-ergic neurons.
Parkinson's and Huntington's Diseases are usually associated with
movement disorders, but often show cognitive impairment (memory loss) as
well.

[0212]Age-Associated Memory Impairment (AAMI) is another age-associated
disorder that is characterized by memory loss in healthy, elderly
individuals in the later decades of life. Crook, T. et al. (1986) Devel.
Neuropsych. 2(4):261-276. Presently, the neural basis for AAMI has not
been precisely defined. However, neuron death with aging has been
reported to occur in many species in brain regions implicated in memory,
including cortex, hippocampus, amygdala, basal ganglia, cholinergic basal
forebrain, locus ceruleus, raphe nuclei, and cerebellum. Crook, T. et al.
(1986) Devel. Neuropsych. 2(4):261-276.

[0213]Vitamin D3 compounds of the invention can protect against
neuron loss by genomic or non-genomic mechanisms. Nuclear vitamin D3
receptors are well known to exist in the periphery but have also been
found in the brain, particularly in the hippocampus and neocortex.
Non-genomic mechanisms may also prevent or retard neuron loss by
regulating intraneuronal and/or peripheral calcium and phosphate levels.
Furthermore, vitamin D3 compounds of the invention may protect
against neuronal loss by acting indirectly, e.g., by modulating serum PTH
levels. For example, a positive correlation has been demonstrated between
serum PTH levels and cognitive decline in Alzheimer's Disease.

[0214]The present method can be performed on cells in culture, e.g. in
vitro or ex vivo, or on cells present in an animal subject, e.g., in
vivo. Vitamin D3 compounds of the invention can be initially tested
in vitro using neurons from embryonic rodent pups (See e.g. U.S. Pat. No.
5,179,109-fetal rat tissue culture), or other mammalian (See e.g. U.S.
Pat. No. 5,089,517-fetal mouse tissue culture) or non-mammalian animal
models. These culture systems have been used to characterize the
protection of peripheral, as well as, central nervous system neurons in
animal or tissue culture models of ischemia, stroke, trauma, nerve crush,
Alzheimer's Disease, Pick's Disease, and Parkinson's Disease, among
others.

[0215]Examples of in vitro systems to study the prevention of destruction
of neocortical neurons include using in vitro cultures of fetal mouse
neurons and glial cells previously exposed to various glutamate agonists,
such as kainate, NMDA, and
α-amino-3-hydroxy-5-methyl-4-isoxazolepronate (AMPA). U.S. Pat. No.
5,089,517. See also U.S. Pat. No. 5,170,109 (treatment of rat
cortical/hippocampal neuron cultures with glutamate prior to treatment
with neuroprotective compound); U.S. Pat. Nos. 5,163,196 and 5,196,421
(neuroprotective excitatory amino acid receptor antagonists inhibit
glycine, kainate, AMPA receptor binding in rats).

[0216]Alternatively, the effects of vitamin D3 compounds of the
invention can be characterized in vivo using animals models. Neuron
deterioration in these model systems is often induced by experimental
trauma or intervention (e.g. application of toxins, nerve crush,
interruption of oxygen supply).

[0217]G. Smooth Muscle Cells

[0218]In yet another aspect, the present invention provides a method of
modulating the activity of a vascular smooth muscle cell by contacting a
vitamin D3-responsive smooth muscle cell with a vitamin D3
compound of the invention to activate or, preferably, inhibit the
activity of the cell. The language "activity of a smooth muscle cell" is
intended to include any activity of a smooth muscle cell, such as
proliferation, migration, adhesion and/or metabolism.

[0219]In certain embodiments, the vitamin D3 compounds of the
invention can be used to treat diseases and conditions associated with
aberrant activity of a vitamin D3-responsive smooth muscle cell. For
example, the present invention can be used in the treatment of
hyperproliferative vascular diseases, such as hypertension induced
vascular remodeling, vascular restenosis and atherosclerosis. In other
embodiments, the compounds of the present invention can be used in
treating disorders characterized by aberrant metabolism of a vitamin
D3-responsive smooth muscle cell, e.g., arterial hypertension.

[0220]The present method can be performed on cells in culture, e.g. in
vitro or ex vivo, or on cells present in an animal subject, e.g., in
vivo. Vitamin D3 compounds of the invention can be initially tested
in vitro as described in Catellot et al. (1982), J. Biol. Chem. 257(19):
11256.

4. Suppression of Renin Expression

[0221]The compounds of the present invention control blood pressure by the
suppression of rennin expression and are useful as antihypertensive
agents Renin-angiotensin regulatory cascade plays a significant role in
the regulation of blood pressure, electrolyte and volume homeostasis (Y.
C. Li, Abstract, DeLuca Symposium on Vitamin D3, Tauc, N. Mex., Jun.
15-Jun. 19, 2002, p. 18). Thus, the invention provides a method of
treating a subject for a vitamin D3 associated state, wherein the
vitamin D3 associated state is a disorder characterized by an
aberrant activity of a cell that expresses renin. The method includes
administering to the subject an effective amount of a compound of formula
I, such that renin expression by the cell is suppressed, and the subject
is thereby treated for hypertension.

5. Bladder Dysfunction

[0222]Morphological bladder changes, including a progressive de-nervation
and hypertrophy of the bladder wall are frequent histological findings in
patients with different bladder disorders leading to overactive bladder
such as bladder disorders associated with, for example, clinical benign
prostatic hyperplasia (BPH) and spinal cord injury.

[0223]The increase in tension and/or strain on the bladder observed in
these conditions has been shown to be associated with cellular and
molecular alterations, e.g., in cytoskeletal and contractile proteins, in
mitochondrial function, and in various enzyme activities of the smooth
muscle cells. The hypertrophy of the bladder wall also involves
alterations in its extracellular matrix and non-smooth muscle components.

[0224]These changes in the bladder are associated with the storage
(irritative) symptoms, in particular frequency, urgency, urge
incontinence and nocturia. These symptoms affect the social,
psychological, domestic, occupational, physical and sexual lives of the
patients leading to a profound negative impact on their quality of life.

[0225]At the present time, an ideal treatment of these symptoms has not
been found. Each of the therapeutic options available (for example,
anti-muscarinics or alpha-blockers) is associated with disadvantages
relating to their mechanism of action, which is based only on the
management of symptoms and not on the treatment of the etiology of the
condition. In fact, the clinical utility of some of the available agents
has been limited by poor efficacy and lack of universal patient
acceptance due to a number of significant side effects.

[0226]As a consequence there is a need for new treatments that provide
improved clinical effectiveness by targeting the underlying etiological
factor, the abnormal growth and consequent dysfunction of bladder smooth
muscle cells.

[0227]As described herein, it has now surprisingly been found that vitamin
D analogs can treat and prevent bladder dysfunction in disorders
associated with bladder hypertrophy, such as bladder overactivity and
clinical BPH. Overactive bladder, also known as detrusor overactivity or
detrusor instability, involves involuntary bladder spasms. A hyperactive
detrusor muscle can cause overactive bladder. Although the underlying
cause of overactive bladder can be neurological disease (e.g., multiple
sclerosis, Parkinson's disease, stroke, spinal cord lesions), nerve
damage caused by abdominal trauma, pelvic trauma, or surgery, stroke,
multiple sclerosis, infection, bladder cancer, drug side effects or
enlarged prostate (BPH), in many cases the cause is idiopathic, i.e. of
unknown cause.

[0228]In addition, such vitamin D related compounds have an application in
the treatment of irritative voiding symptoms associated with BPH. BPH is
associated not only with enlargement of the gland leading to bladder
outlet obstruction (BOO) and symptoms secondary to this, but also to
morphological bladder changes, including a hypertrophy of the bladder
wall and progressive de-nervation. These changes lead to increased
functional demands and disruption of the coordination within the bladder
smooth muscle cells.

6. Uveitis

[0229]Uveitis, a condition comprising inflammation of the eye including
the iris, ciliary body, and choroid, actually comprises a large group of
diverse diseases affecting not only the uvea but also the retina, optic
nerve and vitreous. According to the International Uveitis Study Group,
there are several classifications of uveitis: anterior, intermediate,
posterior and panuveitis (total). Inflammation may be induced by trauma
or toxic or infectious agents, but in most cases the mechanisms seem to
be autoimmune in nature. Symptoms may be acute, sub-acute, chronic
(greater than 3 months duration) and recurrent. The etiology is unknown
in the majority of cases of endogenous uveitis. Uveitis is a major cause
of severe visual impairment. Although the number of patients blinded from
uveitis is unknown, it has been estimated that uveitis accounts for
10-15% of all cases of total blindness in the USA.

A variety of conditions can be described as posterior uveitis: focal,
multifocal or diffuse choroiditis, chorioretinitis, retinochoroiditis,
uveoretinitis or neurouveitis. The condition is usually painless but is
characterised by the presence of floaters, vision impairment (sudden or
gradual) such as blurring of vision, etc., and vision loss. Posterior
uveitis may have several etiologies, and manifests itself in complex and
sometimes misleading clinical conditions. There is growing evidence both
in experimental models and clinically that endogenous posterior
uveoretinitis is often characterised by an exaggerated immune response
which causes tissue destruction. When no apparent infectious or
neoplastic aetiology is found, treatment can be directed towards
dampening the resulting inflammatory cascade and hopefully reducing
tissue damage. In one embodiment, the invention provides a method of
treating uveitis.

7. Interstitial Cystitis

[0230]Interstitial cystitis, referred to herein as "IC", is a chronic
inflammatory bladder disease, also known as chronic pelvic pain syndrome
(CPPS) or painful bladder syndrome (PBS), characterized by pelvic pain,
urinary urgency and frequency. This disease affects maintly females,
although males are also diagnosed with IC. Unlike other bladder
dysfunction conditions, IC is characterized by chronic inflammation of
the bladder wall which is responsible for the symptomatology; in other
words, the cause of the abnormal bladder contractility and chronic pelvic
pain is the chronic inflammation and as a consequence the treatment
should target this etiological component. In fact, the traditional
treatment of bladder dysfunctions, like overactive bladder, with smooth
muscle relaxant agents, is not effective in patients with IC. In one
embodiment, the invention provides a method of treating interstitial
cystitis.

8. Uterine Myomas

[0231]Uterine myomas (also known as uterine leiomyomas/leiomyomata,
fibroids, myomas/myomata, fibromyomas, myofibromas, fibroleiomyomas) are
benign tumours of smooth muscle cells from the uterine myometrium. They
include submucous, subserous and intramural myomas. In one embodiment,
the invention provides a method for the treatment of uterine myomas.

9. Pharmaceutical Compositions

[0232]The invention also provides a pharmaceutical composition, comprising
an effective amount of a vitamin D3 compound of formula I or
otherwise described herein and a pharmaceutically acceptable carrier. In
a further embodiment, the effective amount is effective to treat a
vitamin D3 associated state, as described previously. In an
embodiment, the vitamin D3 compound is administered to the subject
using a pharmaceutically-acceptable formulation, e.g., a
pharmaceutically-acceptable formulation that provides sustained delivery
of the vitamin D3 compound to a subject for at least 12 hours, 24
hours, 36 hours, 48 hours, one week, two weeks, three weeks, or four
weeks after the pharmaceutically-acceptable formulation is administered
to the subject.

[0233]In certain embodiments, these pharmaceutical compositions are
suitable for topical or oral administration to a subject. In other
embodiments, as described in detail below, the pharmaceutical
compositions of the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for the
following: (1) oral administration, for example, drenches (aqueous or
non-aqueous solutions or suspensions), tablets, boluses, powders,
granules, pastes; (2) parenteral administration, for example, by
subcutaneous, intramuscular or intravenous injection as, for example, a
sterile solution or suspension; (3) topical application, for example, as
a cream, ointment or spray applied to the skin; (4) intravaginally or
intrarectally, for example, as a pessary, cream or foam; or (5) aerosol,
for example, as an aqueous aerosol, liposomal preparation or solid
particles containing the compound.

[0234]The phrase "pharmaceutically acceptable" refers to those vitamin
D3 compounds of the present invention, compositions containing such
compounds, and/or dosage forms which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.

[0235]The phrase "pharmaceutically-acceptable carrier" includes
pharmaceutically-acceptable material, composition or vehicle, such as a
liquid or solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting the subject chemical from
one organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,
such as cocoa butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such
as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents,
such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid;
(16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical formulations.

[0236]Wetting agents, emulsifiers and lubricants, such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the compositions.

[0238]Compositions containing a vitamin D3 compound(s) include those
suitable for oral, nasal, topical (including buccal and sublingual),
rectal, vaginal, aerosol and/or parenteral administration. The
compositions may conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host being
treated, the particular mode of administration. The amount of active
ingredient which can be combined with a carrier material to produce a
single dosage form will generally be that amount of the compound which
produces a therapeutic effect. Generally, out of one hundred percent,
this amount will range from about 1 percent to about ninety-nine percent
of active ingredient, preferably from about 5 percent to about 70
percent, most preferably from about 10 percent to about 30 percent.

[0239]Methods of preparing these compositions include the step of bringing
into association a vitamin D3 compound(s) with the carrier and,
optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association a vitamin D3 compound with liquid carriers, or finely
divided solid carriers, or both, and then, if necessary, shaping the
product.

[0240]Compositions of the invention suitable for oral administration may
be in the form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or non-aqueous
liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an
elixir or syrup, or as pastilles (using an inert base, such as gelatin
and glycerin, or sucrose and acacia) and/or as mouth washes and the like,
each containing a predetermined amount of a vitamin D3 compound(s)
as an active ingredient. A compound may also be administered as a bolus,
electuary or paste.

[0241]In solid dosage forms of the invention for oral administration
(capsules, tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more pharmaceutically-acceptable
carriers, such as sodium citrate or dicalcium phosphate, and/or any of
the following: (1) fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,
for example, carboxymethylcellulose, alginates, gelatin, polyvinyl
pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato or
tapioca starch, alginic acid, certain silicates, and sodium carbonate;
(5) solution retarding agents, such as paraffin; (6) absorption
accelerators, such as quaternary ammonium compounds; (7) wetting agents,
such as, for example, acetyl alcohol and glycerol monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such a
talc, calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In
the case of capsules, tablets and pills, the pharmaceutical compositions
may also comprise buffering agents. Solid compositions of a similar type
may also be employed as fillers in soft and hard-filled gelatin capsules
using such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.

[0242]A tablet may be made by compression or molding, optionally with one
or more accessory ingredients. Compressed tablets may be prepared using
binder (for example, gelatin or hydroxypropylmethyl cellulose),
lubricant, inert diluent, preservative, disintegrant (for example, sodium
starch glycolate or cross-linked sodium carboxymethyl cellulose),
surface-active or dispersing agent. Molded tablets may be made by molding
in a suitable machine a mixture of the powdered active ingredient
moistened with an inert liquid diluent.

[0243]The tablets, and other solid dosage forms of the pharmaceutical
compositions of the present invention, such as dragees, capsules, pills
and granules, may optionally be scored or prepared with coatings and
shells, such as enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as to
provide slow or controlled release of the active ingredient therein
using, for example, hydroxypropylmethyl cellulose in varying proportions
to provide the desired release profile, other polymer matrices, liposomes
and/or microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating sterilizing
agents in the form of sterile solid compositions which can be dissolved
in sterile water, or some other sterile injectable medium immediately
before use. These compositions may also optionally contain opacifying
agents and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples of
embedding compositions which can be used include polymeric substances and
waxes. The active ingredient can also be in micro-encapsulated form, if
appropriate, with one or more of the above-described excipients.

[0247]Pharmaceutical compositions of the invention for rectal or vaginal
administration may be presented as a suppository, which may be prepared
by mixing one or more vitamin D3 compound(s) with one or more
suitable nonirritating excipients or carriers comprising, for example,
cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release the
active agent.

[0248]Compositions of the present invention which are suitable for vaginal
administration also include pessaries, tampons, creams, gels, pastes,
foams or spray formulations containing such carriers as are known in the
art to be appropriate.

[0249]Dosage forms for the topical or transdermal administration of a
vitamin D3 compound(s) include powders, sprays, ointments, pastes,
creams, lotions, gels, solutions, patches and inhalants. The active
vitamin D3 compound(s) may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives, buffers,
or propellants which may be required.

[0251]Powders and sprays can contain, in addition to a vitamin D3
compound(s), excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these
substances. Sprays can additionally contain customary propellants, such
as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such
as butane and propane.

[0252]The vitamin D3 compound(s) can be alternatively administered by
aerosol. This is accomplished by preparing an aqueous aerosol, liposomal
preparation or solid particles containing the compound. A nonaqueous
(e.g., fluorocarbon propellant) suspension could be used. Sonic
nebulizers are preferred because they minimize exposing the agent to
shear, which can result in degradation of the compound.

[0253]Ordinarily, an aqueous aerosol is made by formulating an aqueous
solution or suspension of the agent together with conventional
pharmaceutically-acceptable carriers and stabilizers. The carriers and
stabilizers vary with the requirements of the particular compound, but
typically include nonionic surfactants (Tweens, Pluronics, or
polyethylene glycol), innocuous proteins like serum albumin, sorbitan
esters, oleic acid, lecithin, amino acids such as glycine, buffers,
salts, sugars or sugar alcohols. Aerosols generally are prepared from
isotonic solutions.

[0254]Transdermal patches have the added advantage of providing controlled
delivery of a vitamin D3 compound(s) to the body. Such dosage forms
can be made by dissolving or dispersing the agent in the proper medium.
Absorption enhancers can also be used to increase the flux of the active
ingredient across the skin. The rate of such flux can be controlled by
either providing a rate controlling membrane or dispersing the active
ingredient in a polymer matrix or gel-Ophthalmic formulations, eye
ointments, powders, solutions and the like, are also contemplated as
being within the scope of the invention.

[0255]Pharmaceutical compositions of the invention suitable for parenteral
administration comprise one or more vitamin D3 compound(s) in
combination with one or more pharmaceutically-acceptable sterile isotonic
aqueous or nonaqueous solutions, dispersions, suspensions or emulsions,
or sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.

[0256]Examples of suitable aqueous and nonaqueous carriers which may be
employed in the pharmaceutical compositions of the invention include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required particle
size in the case of dispersions, and by the use of surfactants.

[0257]These compositions may also contain adjuvants such as preservatives,
wetting agents, emulsifying agents and dispersing agents. Prevention of
the action of microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol,
phenol sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into the
compositions. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents which
delay absorption such as aluminum monostearate and gelatin.

[0258]In some cases, in order to prolong the effect of a drug, it is
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a liquid
suspension of crystalline or amorphous material having poor water
solubility. The rate of absorption of the drug then depends upon its rate
of dissolution which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug form is accomplished by dissolving or
suspending the drug in an oil vehicle.

[0259]Injectable depot forms are made by forming microencapsule matrices
of vitamin D3 compound(s) in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to polymer, and
the nature of the particular polymer employed, the rate of drug release
can be controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable formulations are
also prepared by entrapping the drug in liposomes OT microemulsions which
are compatible with body tissue.

[0260]When the vitamin D3 compound(s) are administered as
pharmaceuticals, to humans and animals, they can be given per se or as a
pharmaceutical composition containing, for example, 0.1 to 99.5% (more
preferably, 0.5 to 90%) of active ingredient in combination with a
pharmaceutically-acceptable carrier.

[0261]Regardless of the route of administration selected, the vitamin
D3 compound(s), which may be used in a suitable hydrated form,
and/or the pharmaceutical compositions of the present invention, are
formulated into pharmaceutically-acceptable dosage forms by conventional
methods known to those of skill in the art.

[0262]Actual dosage levels and time course of administration of the active
ingredients in the pharmaceutical compositions of the invention may be
varied so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being toxic to
the patient. An exemplary dose range is from 0.1 to 10 mg per day.

[0263]A preferred dose of the vitamin D3 compound for the present
invention is the maximum that a patient can tolerate and not develop
serious hypercalcemia. Preferably, the vitamin D3 compound of the
present invention is administered at a concentration of about 0.001 μg
to about 100 μg per kilogram of body weight, about 0.001-about 10
μg/kg or about 0.001 μg-about 100 μg/kg of body weight. Ranges
intermediate to the above-recited values are also intended to be part of
the invention.

Exemplification of the Invention

[0264]The invention is further illustrated by the following examples which
should in no way should be construed as being further limiting.

Synthesis of Compounds of the Invention

EXPERIMENTAL

[0265]All operations involving vitamin D3 analogs were conducted in
amber-colored glassware in a nitrogen atmosphere. Tetrahydrofuran was
distilled from sodium-benzophenone ketyl just prior to its use and
solutions of solutes were dried with sodium sulfate. Melting points were
determined on a Thomas-Hoover capillary apparatus and are uncorrected.
Optical rotations were measured at 25° C. 1H NMR spectra were
recorded at 400 MHz in CDCl3 unless indicated otherwise. TLC was
carried out on silica gel plates (Merck PF-254) with visualization under
short-wavelength UV light or by spraying the plates with 10%
phosphomolybdic acid in methanol followed by heating. Flash
chromatography was carried out on 40-65 μm mesh silica gel.
Preparative HPLC was performed on a 5×50 cm column and 15-30 μm
mesh silica gel at a flow rate of 100 ml/min. The results are summarized
in Tables 1 and 2 fox compounds 1-14.

[0330]The maximum tolerated dose of the vitamin D3 compounds of the
invention were determined in eight week-old female C57BL/6 mice (3
mice/group) dosed orally (0.1 ml/mouse) with various concentrations of
Vitamin D3 analogs daily for four days. Analogs were formulated in
miglyol for a final concentration of 0.01, 0.03, 0.1 0.3, 1, 3, 10, 30,
100 and 300 μg/kg when given at 0.1 ml/mouse p.o. daily. Blood for
serum calcium assay was drawn by tail bleed on day five, the final day of
the study. Serum calcium levels were determined using a colorimetric
assay (Sigma Diagnostics, procedure no. 597). The highest dose of analog
tolerated without inducing hypercalcemia (serum calcium >10.7 mg/dl)
was taken as the maximum tolerated dose (MTD). Table 3 shows the relative
MTD for compounds (1)-(14).

[0332]Briefly, peripheral blood mononuclear cells (PBMC) were separated
from buffy coats by Ficoll gradient and the same number
(3×105) of allogeneic PBMC from 2 different donors were
co-cultured in 96-well flat-bottom plates. The vitamin D3 compounds
were added to each of the cultures. After 5 days, IFN-γ production
in the MLR assay was measured by ELISA and the results expressed as
amount (nM) of test compound required to induce 50% inhibition of
IFN-γ production (IC50). The results are summarized in Table
3.

The activity of Calcitriol and Vitamin D3 Analogues on the Growth and
Function of Bladder Cells

[0334]The Inventors' finding that calcitriol and Vitamin D3 analogues
can have an effect on the growth and function of bladder cells has been
proven in in vitro models by culturing human stromal bladder cells. The
Inventors confirmed the presence of vitamin D receptors (VDRs), as
previously reported in the literature, on these cells (see below in FIG.
1).

[0335]In these models, calcitriol (the activated form of vitamin D3)
and other vitamin D3 analogues (compounds (4), (6), (8) and (10)
have been shown to be effective in inhibiting the basal (FIG. 2) growth
of bladder cells. This activity, never reported before, is dose dependent
with an IC50 of 9.8±7×10-15 for calcitriol
(1,25-dihydroxycholecalciferol) (on basal cells).

[0336]A similar investigation was performed on a number of other vitamin D
compounds and the results (expressed as -Log IC50) are shown in the
table below. Data in the table refers to inhibitors effect of the
compound on basal human bladder cell growth in cells which are not
stimulated with testosterone or (in one case) are stimulated. The maximum
tolerated dose (MTD) in rats is also listed for each compound (Table 5).

[0338]Compounds were evaluated calculating a number of physicochemical and
structural properties related to druggability, based on their
bidimensional structures. The ACD/labs software (v. 7.0, Advanced
Chemistry Development Inc., Toronto, Canada) was used. The calculated
physicochemical properties included: the octanol/water partition
coefficients in logarithmic scale (ACDlogP), the octanol/water
distribution coefficients at pH 7.4 in logarithmic scale (logD7.4) and
the molar solubility at pH 7.4 logarithmic transformed (logS7.4). The
calculated structural properties included: molecular weight (MW), molar
volume (expressed as cm3), molar refractivity (expressed as
cm3), number of hydrogen bond donors and acceptors (i.e. HDonors and
HAcceptors), number of freely rotatable bonds (FRB), number of violations
to Lipinski rules and polar surface area (PSA expresses as Å2).
Results can be found at Table 7.

[0339]To evaluate the intestinal absorption potential of selected
compounds, the maximum absorbable dose (MAD) in mouse, rat and human
intestines was also calculated using a modification of the original
equation (Johnson 1996, Hilgers 2003): MAD (mg)=SPe(A/ILV)SIVSITT (Eq.
1), where S is the solubility measured at pH 7.4 (mg/ml), Pe is the
permeability measured in artificial membranes (PAMPA) or in the apical to
basolateral direction of Caco-2 cells (cm/sec), A is the intestinal
surface area (cm2), ILV is the intestinal lumen volume (cm3),
SIV is the small intestinal volume (ml) and SITT is the small intestinal
transit time (sec). Results are found at Table 8.

[0340]The following in vitro tests were applied to characterize the
compounds:

[0341]Solubility at pH 7. A 96-well plate format assay was used. The
compound stock solution was diluted at a concentration of 10 μM in
aqueous buffers at a pH value of 7. Solutions were filtered through a
0.22 μm and concentrations of the concentration of the compound in the
filtrate was determined using LC-MS/MS in comparison with 1 and 10 μM
standards. The measurements were expressed as mM.

[0342]Metabolic Stability (hCYP34A4). The relative stability of the
substrate was determined by measuring the amount of substrate remaining
following incubation with human cDNA expressed CYP3A4 microsomal
preparations (Gentest, 6 pmol) against a control microsomal incubation
containing no active cytochrome P450. The assay was performed in a
96-well plate format. Each compound was incubated at a concentration of 2
μM for 60 min at 37° C. LC-MS/MS was used for determining the
compound remaining after incubation. The results were expressed as %
remaining.

[0343]Permeability by Passive Diffusion (PAMPA). Experiments were
performed in 96-well acceptor and donor plates using 15% soy lecithin in
n-dodecane artificial membranes. The acceptor plate (96 well hydrophobic
filter plate (MAIP N45, Millipore)) was prepared by adding 4 μL of
artificial membrane material on the top of the filter and the plate was
filled with 200 μL of HEPES buffered HBSS (pH 7.4). The donor plate
(an indented 96-well plate from p-ION, MA) was filled with 200 μL of
HEPES buffered HBSS (pH 7.4) containing 10 μM of the test compounds.
The acceptor plate was placed onto the donor plate to form a "sandwich"
and was incubated at 37° C. for 4 hours. After the incubation
period, acceptor, donor and initial donor solution (reference) were
analysed via LC-MS/MS. Data were reported as bilateral Peff in
cm×10-6/sec and % retention in the membrane.

[0344]Apparent Permeability on Caco-2 cells. Human colon adenocarcinoma
(Caco-2) cells were obtained from the American Type Culture Collection
(Rockville, Md.). Permeability studies were performed using a 24-well
format in both transport directions, apical to basolateral (A→B)
and basolateral to apical (B→A), on Caco-2 monolayers. Fresh donor
solution containing 10 μM test compound was added to either the apical
or the basolateral side, while drug-free medium was placed on the
opposite side. The 24-transwell plates were placed on a plate shaker at
37° C. After 2 h, the buffer from the receiving and donor chambers
were collected and aliquots were analysed via LC-MS/MS. The data reported
were the permeability cm10-6/sec and the efflux ratio. Results can
be found at Table 9.

[0346]1. BHT and BHA is suspended in Miglyol 812 and warmed to about
50° C. with stirring, until dissolved.

[0347]2. 1,25-Dihydroxy-16-ene-23-yne-20-cyclopyl-cholecalciferol is
dissolved in the solution from step 1 at 50° C. [0348]3. The
solution from Step 2 is cooled at room temperature. [0349]4. The solution
from Step 3 is filled into soft gelatin capsules.Note: All manufacturing
steps are performed under a nitrogen atmosphere and protected from light.

[0351]1. Di-α-Tocopherol is suspended in Miglyol 812 and warmed to
about 50° C. with stirring, until dissolved.

[0352]2. 1,25-Dihydroxy-16-ene-23-yne-20-cyclopyl-cholecalciferol is
dissolved in the solution from step 1 at 50° C.

[0353]3. The solution from Step 2 is cooled at room temperature.

[0354]4. The solution from Step 3 is filled into soft gelatin capsules.

Incorporation by Reference

[0355]The contents of all references (including literature references,
issued patents, published patent applications, and co-pending patent
applications) cited throughout this application are hereby expressly
incorporated herein in their entireties by reference.

EQUIVALENTS

[0356]Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents of the
specific embodiments of the invention described herein. Such equivalents
are intended to be encompassed by the following claims.